A Recombinant Vesicular Stomatitis Virus Ebola Vaccine.

BACKGROUND: The worst Ebola virus disease (EVD) outbreak in history has resulted in more than 28,000 cases and 11,000 deaths. We present the final results of two phase 1 trials of an attenuated, replication-competent, recombinant vesicular stomatitis virus (rVSV)-based vaccine candidate designed to prevent EVD. METHODS: We conducted two phase 1, placebo-controlled, double-blind, dose-escalation trials of an rVSV-based vaccine candidate expressing the glycoprotein of a Zaire strain of Ebola virus (ZEBOV). A total of 39 adults at each site (78 participants in all) were consecutively enrolled into groups of 13. At each site, volunteers received one of three doses of the rVSV-ZEBOV vaccine (3 million plaque-forming units [PFU], 20 million PFU, or 100 million PFU) or placebo. Volunteers at one of the sites received a second dose at day 28. Safety and immunogenicity were assessed. RESULTS: The most common adverse events were injection-site pain, fatigue, myalgia, and headache. Transient rVSV viremia was noted in all the vaccine recipients after dose 1. The rates of adverse events and viremia were lower after the second dose than after the first dose. By day 28, all the vaccine recipients had seroconversion as assessed by an enzyme-linked immunosorbent assay (ELISA) against the glycoprotein of the ZEBOV-Kikwit strain. At day 28, geometric mean titers of antibodies against ZEBOV glycoprotein were higher in the groups that received 20 million PFU or 100 million PFU than in the group that received 3 million PFU, as assessed by ELISA and by pseudovirion neutralization assay. A second dose at 28 days after dose 1 significantly increased antibody titers at day 56, but the effect was diminished at 6 months. CONCLUSIONS: This Ebola vaccine candidate elicited anti-Ebola antibody responses. After vaccination, rVSV viremia occurred frequently but was transient. These results support further evaluation of the vaccine dose of 20 million PFU for preexposure prophylaxis and suggest that a second dose may boost antibody responses. (Funded by the National Institutes of Health and others; rVSV∆G-ZEBOV-GP ClinicalTrials.gov numbers, NCT02269423 and NCT02280408 .).

Comparative evaluation of pathogenicity of three isolates of vesicular stomatitis virus (Indiana serotype) in pigs.

Vesicular stomatitis (VS) is a notifiable disease of livestock affecting cattle, horses, pigs and humans. Vesicular stomatitis virus (VSV) serotypes Indiana and New Jersey are endemic to Central America; however, they also cause sporadic and scattered outbreaks in various countries in South and North America, including the USA. In order to develop an effective experimental challenge model for VSV, we compared the pathogenicity of three VSV serotype Indiana isolates in 36 4-5 week-old pigs. Two bovine isolates of Central American origin and one equine isolate from the USA were used for the experimental infections. Each pig was inoculated with a single isolate by both the intradermal and intranasal routes. Clinical signs of VSV infection were recorded daily for 10 days post-inoculation (days p.i.). Nasal and tonsillar swab samples and blood were collected to monitor immune responses, virus replication and shedding. Post-challenge, characteristic signs of VS were observed, including vesicles on the nasal planum and coronary bands, lameness, loss of hoof walls and pyrexia. Pigs inoculated with the Central American isolates showed consistently more severe clinical signs in comparison to the pigs infected with the USA isolate. Genomic RNA was isolated from the original challenge virus stocks, sequenced and compared to VSV genomes available in GenBank. Comparative genome analysis demonstrated significant differences between the VSV isolate from the USA and the two Central American isolates. Our results indicate that the Central American isolates of VSV serotype Indiana used in this study are more virulent in swine than the USA VSV serotype Indiana isolate and represent good candidate challenge strains for future VSV studies.

High dose of vesicular stomatitis virus-vectored Ebola virus vaccine causes vesicular disease in swine without horizontal transmission.

ABSTRACTThe recent impact of Ebola virus disease (EVD) on public health in Africa clearly demonstrates the need for a safe and efficacious vaccine to control outbreaks and mitigate its threat to global health. ERVEBO® is an effective recombinant Vesicular Stomatitis Virus (VSV)-vectored Ebola virus vaccine (VSV-EBOV) that was approved by the FDA and EMA in late 2019 for use in prevention of EVD. Since the parental virus VSV, which was used to construct VSV-EBOV, is pathogenic for livestock and the vaccine virus may be shed at low levels by vaccinated humans, widespread deployment of the vaccine requires investigation into its infectivity and transmissibility in VSV-susceptible livestock species. We therefore performed a comprehensive clinical analysis of the VSV-EBOV vaccine virus in swine to determine its infectivity and potential for transmission. A high dose of VSV-EBOV resulted in VSV-like clinical signs in swine, with a proportion of pigs developing ulcerative vesicular lesions at the nasal injection site and feet. Uninoculated contact control pigs co-mingled with VSV-EBOV-inoculated pigs did not become infected or display any clinical signs of disease, indicating the vaccine is not readily transmissible to naïve pigs during prolonged close contact. In contrast, virulent wild-type VSV Indiana had a shorter incubation period and was transmitted to contact control pigs. These results indicate that the VSV-EBOV vaccine causes vesicular illness in swine when administered at a high dose. Moreover, the study demonstrates the VSV-EBOV vaccine is not readily transmitted to uninfected pigs, encouraging its safe use as an effective human vaccine.

rVSVΔG-ZEBOV-GP (also designated V920) recombinant vesicular stomatitis virus pseudotyped with Ebola Zaire Glycoprotein: Standardized template with key considerations for a risk/benefit assessment.

The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) was formed to evaluate the safety and characteristics of live, recombinant viral vector vaccines. A recent publication by the V3SWG described live, attenuated, recombinant vesicular stomatitis virus (rVSV) as a chimeric virus vaccine for HIV-1 (Clarke et al., 2016). The rVSV vector system is being explored as a platform for development of multiple vaccines. This paper reviews the molecular and biological features of the rVSV vector system, followed by a template with details on the safety and characteristics of a rVSV vaccine against Zaire ebolavirus (ZEBOV). The rVSV-ZEBOV vaccine is a live, replication competent vector in which the VSV glycoprotein (G) gene is replaced with the glycoprotein (GP) gene of ZEBOV. Multiple copies of GP are expressed and assembled into the viral envelope responsible for inducing protective immunity. The vaccine (designated V920) was originally constructed by the National Microbiology Laboratory, Public Health Agency of Canada, further developed by NewLink Genetics Corp. and Merck & Co., and is now in final stages of registration by Merck. The vaccine is attenuated by deletion of the principal virulence factor of VSV (the G protein), which also removes the primary target for anti-vector immunity. The V920 vaccine caused no toxicities after intramuscular (IM) or intracranial injection of nonhuman primates and no reproductive or developmental toxicity in a rat model. In multiple studies, cynomolgus macaques immunized IM with a wide range of virus doses rapidly developed ZEBOV-specific antibodies measured in IgG ELISA and neutralization assays and were fully protected against lethal challenge with ZEBOV virus. Over 20,000 people have received the vaccine in clinical trials; the vaccine has proven to be safe and well tolerated. During the first few days after vaccination, many vaccinees experience a mild acute-phase reaction with fever, headache, myalgia, and arthralgia of short duration; this period is associated with a low-level viremia, activation of anti-viral genes, and increased levels of chemokines and cytokines. Oligoarthritis and rash appearing in the second week occur at a low incidence, and are typically mild-moderate in severity and self-limited. V920 vaccine was used in a Phase III efficacy trial during the West African Ebola epidemic in 2015, showing 100% protection against Ebola Virus Disease, and it has subsequently been deployed for emergency control of Ebola outbreaks in central Africa. The template provided here provides a comprehensive picture of the first rVSV vector to reach the final stage of development and to provide a solution to control of an alarming human disease.

Regulation of the C5a receptor promoter in glial cells: Minimal dependence upon the CCAAT element in astrocytes.

The cleavage product of C5, C5a, is an important anaphylatoxin. This inflammatory mediator exerts its effects by binding to the C5a receptor (C5aR, CD88), a member of the seven transmembrane-spanning G protein-coupled receptor family. Recent evidence has suggested that C5aR is expressed in diverse cell types including myeloid cells, endothelium and parenchymal cells in many tissues. Some data have suggested a role for C5a in neuroinflammation, however the molecular mechanisms responsible for C5aR expression in glial cells are largely unknown. In this report, we demonstrate higher levels of C5aR transcription in microglia compared to astrocytes. NF-YA protein from microglial nuclear extracts forms strong complexes with the C5aR CCAAT motif, suggesting regulation similar to that previously described in macrophages. In astrocytes, there is weak protein binding at the CCAAT box and reporter gene assays suggest minimal dependence upon this site for transcriptional regulation in primary astrocytes. Instead, there are several sites that exhibit some level of transcriptional control and the minimal construct directs significant promoter activity. These data suggest that C5aR transcriptional control in astrocytes is distinct from regulation in myeloid cells.

Transcriptional control of the C3a receptor gene in glial cells: Dependence upon AP-1 but not Ets.

The C3a anaphylatoxin has been implicated in several autoimmune states including arthritis and multiple sclerosis. The expression pattern of the C3a receptor (C3aR) is critically important in C3a biology, yet very little is known about the transcriptional control of the C3aR gene. Since C3a is hypothesized to play a role in neuroinflammation, we investigated the molecular mechanisms governing C3aR expression in astrocytes and microglia. In the current study, we demonstrate that C3aR transcription in microglia mirrors that in other macrophages, with strong transcription factor binding at the AP-1 and Ets sites. In transformed astrocytes there is evidence for AP-1 and Ets binding in the C3aR promoter region, while in primary astrocytes these sites do not apparently bind strongly to these transcription factors. Primary astrocytes lack a strong complex at the C3aR AP-1 site and reporter gene assays indicate a much smaller contribution of this site to transcriptional activity. Although EMSA analyses using astrocyte extracts show strong complexes exist at the Ets site, this sequence has a minimal activity in reporter assays. Finally, in vivo footprinting demonstrates much stronger DNA binding activity at both the AP-1 and Ets sites in microglia when compared to astrocytes. Collectively, our data demonstrate that transcriptional control of C3aR expression in astrocytes is fundamentally different than that in myeloid cells.

Transcriptional control of complement receptor gene expression.

Immune complement is a critical system in the immune response and protection of host cells from damage by complement is critical during inflammation. The expression of the receptors for the inflammatory anaphylatoxin molecules is also key in immunity. In order to fully appreciate the biology of complement, a basic understanding of the molecular regulation of complement receptor gene expression is critical, yet these kinds of studies are lacking for many genes. Importantly, recent genetic studies have demonstrated that promoter-enhancer polymorphisms can contribute to pathology in diseases such as atypical hemolytic uremic syndrome. This review will focus on what is currently known about the genetic regulation of key protective complement receptors genes including CR1 (CD35), CR2 (CD21), Crry, MCP (CD46), DAF (CD55), and CD59. In addition, the regulation of the anaphylatoxin receptors genes, C3aR and C5aR (CD88) will also be discussed. Since new research continuously uncovers novel functions for these proteins, a greater appreciation of the mechanisms involved in gene regulation will be critical for understanding the biology of these molecules.

Addition of αGal HyperAcute™ technology to recombinant avian influenza vaccines induces strong low-dose antibody responses.

Highly pathogenic avian influenza represents a severe public health threat. Over the last decade, the demand for highly efficacious vaccines against avian influenza viruses has grown, especially after the 2013 H7N9 outbreak in China that resulted in over 600 human cases with over 200 deaths. Currently, there are several H5N1 and H7N9 influenza vaccines in clinical trials, all of which employ traditional oil-in-water adjuvants due to the poor immunogenicity of avian influenza virus antigens. In this study, we developed potent recombinant avian influenza vaccine candidates using HyperAcute™ Technology, which takes advantage of naturally-acquired anti-αGal immunity in humans. We successfully generated αGal-positive recombinant protein and virus-like particle vaccine candidates of H5N1 and H7N9 influenza strains using either biological or our novel CarboLink chemical αGal modification techniques. Strikingly, two doses of 100 ng αGal-modified vaccine, with no traditional adjuvant, was able to induce a much stronger humoral response in αGT BALB/c knockout mice (the only experimental system readily available for testing αGal in vivo) than unmodified vaccines even at 10-fold higher dose (1000 ng/dose). Our data strongly suggest that αGal modification significantly enhances the humoral immunogenicity of the recombinant influenza vaccine candidates. Use of αGal HyperAcute™ technology allows significant dose-sparing while retaining desired immunogenicity. Our success in the development of highly potent H5N1 and H7N9 vaccine candidates demonstrated the potential of αGal HyperAcute™ technology for the development of vaccines against other infectious diseases.

Safety and immunogenicity of the rVSV∆G-ZEBOV-GP Ebola virus vaccine candidate in healthy adults: a phase 1b randomised, multicentre, double-blind, placebo-controlled, dose-response study.

BACKGROUND: The 2014 Zaire Ebola virus outbreak highlighted the need for a safe, effective vaccine with a rapid onset of protection. We report the safety and immunogenicity of the recombinant vesicular stomatitis virus-Zaire Ebola virus envelope glycoprotein vaccine (rVSV∆G-ZEBOV-GP) across a 6 log10 dose range in two sequential cohorts. METHODS: In this phase 1b double-blind, placebo-controlled, dose-response study we enrolled and randomly assigned healthy adults (aged 18-61 years) at eight study sites in the USA to receive a single injection of vaccine or placebo, administered by intramuscular injection. In cohort 1, participants were assigned to receive 3 × 103, 3 × 104, 3 × 105, or 3 × 106 PFU doses of rVSV∆G-ZEBOV-GP or placebo. In cohort 2, participants were assigned to receive 3 × 106, 9 × 106, 2 × 107, or 1 × 108 PFU doses of rVSV∆G-ZEBOV-GP or placebo. Participants were centrally allocated by the study statistician to vaccine groups or placebo through computer-generated randomisation lists. The primary safety outcome was incidence of adverse events within 14 days in the modified intention-to-treat population (all randomly assigned participants who received vaccine or placebo), and the primary outcome for immunogenicity was IgG ELISA antibody titres at day 28 in the per-protocol population. Surveillance was enhanced for arthritis and dermatitis through to day 56. This study is registered with ClinicalTrials.gov, number NCT02314923. FINDINGS: Between Dec 26, 2014, and June 8, 2015, 513 participants were enrolled and randomly assigned; one was not immunised because of unsuccessful phlebotomy. In cohort 1, 256 participants received vaccine (3 × 103 [n=64], 3 × 104 [n=64], 3 × 105 [n=64], or 3 × 106 PFU [n=64]) and 74 received placebo. In cohort 2, 162 participants received vaccine (3 × 106 [n=20], 9 × 106 [n=47], 2 × 107 [n=47], or 1 × 108 PFU [n=48]) and 20 received placebo. Most adverse events occurred in the first day after vaccination, and were mild to moderate in intensity, of a short duration, and more frequent at high vaccine doses (9 × 106 PFU and greater). At the 2 × 107 PFU dose (used in phase 3 trials), the most common local adverse events versus placebo within the first 14 days were arm pain (57·4% [27 of 47] vs 7·4% [seven of 94]) and local tenderness (59·6% [28 of 47] vs 8·5% [eight of 94]). The most common systemic adverse events at the 2 × 107 PFU dose versus placebo, occurring in the first 14 days, were headache (46·8% [22 of 47] vs 27·7% [26 of 94]), fatigue (38·3% [18 of 47] vs 19·1% [18 of 94]), myalgia (34·0% [16 of 47] vs 10·6% [10 of 94]), subjective fever (29·8% [14 of 47] vs 2·1% [two of 94]), shivering or chills (27·7% [13 of 47] vs 7·4% [seven of 94]), sweats (23·4% [11 of 47] vs 3·2% [three of 94]), joint aches and pain (19·1% [nine of 47] vs 7·4% [seven of 94]), objective fever (14·9% [seven of 47] vs 1·1% [one of 94]), and joint tenderness or swelling (14·9% [seven of 47] vs 2·1% [two of 94]). Self-limited, post-vaccination arthritis occurred in 4·5% (19 of 418) of vaccinees (median onset 12·0 days [IQR 10-14]; median duration 8·0 days [6-15]) versus 3·2% (three of 94) of controls (median onset 15·0 days [6-20]; median duration 47·0 days [37-339]), with no apparent dose relationship. Post-vaccination dermatitis occurred in 5·7% (24 of 418) of vaccinees (median onset 9·0 days [IQR 2-12]; median duration 7·0 days [4-9]) versus 3·2% (three of 94) of controls (median onset 5·0 days [3-53]; median duration 33·0 days [5-370]). A low-level, transient, dose-dependent viraemia occurred in concert with early reactogenicity. Antibody responses were observed in most participants by day 14. IgG and neutralising antibody titres were dose-related (p=0·0003 for IgG ELISA and p<0·0001 for the 60% plaque-reduction neutralisation test [PRNT60] by linear trend). On day 28 at the 2 × 107 PFU dose, the geometric mean IgG ELISA endpoint titre was 1624 (95% CI 1146-2302) and seroconversion was 95·7% (95% CI 85·5-98·8); the geometric mean neutralising antibody titre by PRNT60 was 250 (176-355) and seroconversion was 95·7% (85·5-98·8). These robust immunological responses were sustained for 1 year. INTERPRETATION: rVSV∆G-ZEBOV-GP was well tolerated and stimulated a rapid onset of binding and neutralising antibodies, which were maintained through to day 360. The immunogenicity results support selection of the 2 × 107 PFU dose. FUNDING: Biomedical Advanced Research and Development Authority, US Department of Health and Human Services.

Transcriptional regulation of the murine C5a receptor gene: NF-Y is required for basal and LPS induced expression in macrophages and endothelial cells.

The anaphylatoxin receptors of the complement system are important in immune defense but also play a role in autoimmune disease. Reports have demonstrated induced C5a receptor (C5aR) expression in a number of disease states, yet little is known about the regulation of this gene. We have examined sequences in the presumptive promoter-enhancer region in order to study the regulation of this gene. Rapid amplification of cDNA ends (RACE) analyses were used to identify the transcriptional start site, and we then cloned 2278 bp of sequence from this region for use in luciferase assays. Deletion analyses of 5' sequences demonstrated that the majority of this region is dispensable for expression in macrophages and endothelial cells (ECs). A 232 bp region proximal to the transcription start site was fully capable of directing expression in macrophages and ECs, while being minimally active in cells that do not express the receptor. The transcriptional regulatory site most critical for this expression matches the consensus sequence for nuclear factor-Y (NF-Y) at position -96. Site-directed mutagenesis of this site resulted in a 70-90% decrease in luciferase activity depending on the cell type. Electrophoretic mobility shift/supershift assay (EMSA) analyses demonstrated the specific binding of NF-Y to labeled oligonucleotides containing the putative CCAAT site with macrophages and EC nuclear extracts, and antibodies to NF-Y were able to supershift this -96 NF-Y complex. We also demonstrate LPS leads to enhanced C5aR transcription and this is mediated predominantly through the NF-Y site. The data reported in this study might be critical for determination of transcription factors that can be targeted pharmacologically to modulate the expression of the C5aR in infectious disease or autoimmunity.

Expression of C5a in the brain does not exacerbate experimental autoimmune encephalomyelitis.

Complement is implicated in the pathology of neurodegenerative and inflammatory disease in the central nervous system (CNS). Although studies demonstrate that inhibition of complement activation attenuates disease development in the CNS, the specific complement components that contribute to the pathogenesis of CNS diseases remain unclear. To dissect the role of C5a in CNS disease, we developed a transgenic mouse that produces C5a exclusively in the brain using the astrocyte-specific, murine glial fibrillary acidic protein (GFAP) promoter. C5a/GFAP mice develop normally and do not demonstrate any signs of spontaneous inflammation or neurodegeneration with age. Using C5a/GFAP mice, we examined the outcome of the animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). To our surprise the onset and severity of myelin oligodendrocyte glycoprotein-induced EAE was essentially identical between C5a/GFAP and control mice. These results demonstrate that C5a, despite it is pro-inflammatory functions, is not critical to the development and progression of EAE.

c-Jun and c-Fos regulate the complement factor H promoter in murine astrocytes.

The complement system is a critical component of innate immunity that requires regulation to avoid inappropriate activation. This regulation is provided by many proteins, including complement factor H (CFH), a critical regulator of the alternative pathway of complement activation. Given its regulatory function, mutations in CFH have been implicated in diseases such as age-related macular degeneration and membranoproliferative glomerulonephritis, and central nervous system diseases such as Alzheimer's disease, Parkinson's disease, and a demyelinating murine model, experimental autoimmune encephalomyelitis (EAE). There have been few investigations on the transcriptional regulation of CFH in the brain and CNS. Our studies show that CFH mRNA is present in several CNS cell types. The murine CFH (mCFH) promoter was cloned and examined through truncation constructs and we show that specific regions throughout the promoter contain enhancers and repressors that are positively regulated by inflammatory cytokines in astrocytes. Database mining of these regions indicated transcription factor binding sites conserved between different species, which led to the investigation of specific transcription factor binding interactions in a 241 base pair (bp) region at -416 bp to -175 bp that showed the strongest activity. Through supershift analysis, it was determined that c-Jun and c-Fos interact with the CFH promoter in astrocytes in this region. These results suggest a relationship between cell cycle and complement regulation, and how these transcription factors and CFH affect disease will be a valuable area of investigation.

Transcriptional control of genes for soluble complement cascade regulatory proteins.

The complement cascade of the immune system is an important mediator of the inflammatory response to infection; however it is crucial that this pathway is tightly regulated to prevent uncontrolled activation, which can lead to damage to host tissues. The complement system has many regulators that control activation; both membrane-bound and soluble factors. This review will focus on what is currently known about the transcriptional regulation of the soluble complement regulatory genes C1-inhibitor, complement factor I, complement factor H and C4-binding protein. The absence or mutation of these regulators is all associated with specific disease, and yet their contribution to disease is often poorly understood. It is through full understanding of these genes that we can comprehend the diseases with which they are implicated, and thus prove why knowledge of the transcriptional regulation of these genes is valuable.

CNS-specific expression of C3a and C5a exacerbate demyelination severity in the cuprizone model.

Demyelination in the central nervous system (CNS) is known to involve several immune effector mechanisms, including complement proteins. Local production of complement by glial cells in the brain can be both harmful and protective. To investigate the roles of C3a and C5a in demyelination and remyelination pathology we utilized the cuprizone model. Transgenic mice expressing C3a or C5a under the control of the glial fibrillary acidic protein (GFAP) promoter had exacerbated demyelination and slightly delayed remyelination in the corpus callosum compared to WT mice. C3a and C5a transgenic mice had increased cellularity in the corpus callosum due to increase activation and/or migration of microglia. Oligodendrocytes migrated to the corpus callosum in higher numbers during early remyelination events in C3a and C5a transgenic mice, thus enabling these mice to remyelinate as effectively as WT mice by the end of the 10 week study. To determine the effects of C3a and/or C5a on individual glial subsets, we created murine recombinant C3a and C5a proteins. When microglia and mixed glial cultures were stimulated with C3a and/or C5a, we observed an increase in the production of proinflammatory cytokines and chemokines. In contrast, astrocytes had decreased cytokine and chemokine production in the presence of C3a and/or C5a. We also found that the MAPK pathway proteins JNK and ERK1/2 were activated in glia upon stimulation with C3a and C5a. Overall, our findings show that although C3a and C5a production in the brain play a negative role during demyelination, these proteins may aid in remyelination.

Novel suspension cell-based vaccine production systems for Rift Valley fever virus-like particles.

Rift Valley fever virus (RVFV) is an arthropod-borne pathogen that often results in severe morbidity and mortality in both humans and livestock. As its geographic range continues to expand, it presents a real threat to naïve populations around the world by accidental introduction (e.g., the result of increased travel) or intentional release (e.g., a bioterror event). While there is a clear need for a safe and efficacious vaccine against this emerging and re-emerging pathogen, no FDA-approved vaccine is currently available. This need was addressed by the establishment of novel mammalian and insect suspension cell line systems for the efficient production of RVF virus-like particle (VLP)-based vaccine candidates. A direct comparison of the production of RVF VLPs in these systems was performed. Optimization and characterization resulted in a production platform suitable for scale-up. Furthermore, RVF VLP-based vaccines were tested in a lethal challenge model and showed full protection, demonstrating that RVF VLPs present promising RVFV vaccine candidates.

A replication-incompetent Rift Valley fever vaccine: chimeric virus-like particles protect mice and rats against lethal challenge.

Virus-like particles (VLPs) present viral antigens in a native conformation and are effectively recognized by the immune system and therefore are considered as suitable and safe vaccine candidates against many viral diseases. Here we demonstrate that chimeric VLPs containing Rift Valley fever virus (RVFV) glycoproteins G(N) and G(C), nucleoprotein N and the gag protein of Moloney murine leukemia virus represent an effective vaccine candidate against Rift Valley fever, a deadly disease in humans and livestock. Long-lasting humoral and cellular immune responses are demonstrated in a mouse model by the analysis of neutralizing antibody titers and cytokine secretion profiles. Vaccine efficacy studies were performed in mouse and rat lethal challenge models resulting in high protection rates. Taken together, these results demonstrate that replication-incompetent chimeric RVF VLPs are an efficient RVFV vaccine candidate.

Astrocyte-specific expression of a soluble form of the murine complement control protein Crry confers demyelination protection in the cuprizone model.

Complement has been implicated as a potential effector mechanism in neurodegeneration; yet the precise role of complement in this process remains elusive. In this report, we have utilized the cuprizone model of demyelination-remyelination to examine the contribution of complement to disease. C1q deposition was observed in the corpus callosum of C57BL/6 mice during demyelination, suggesting complement activation by apoptotic oligodendrocyte debris. Simultaneously, these mice lost expression of the rodent complement regulatory protein, Crry. A soluble CNS-specific form of the Crry protein (sCrry) expressed in a transgenic mouse under the control of an astrocyte-specific promoter was induced in the corpus callosum during cuprizone treatment. Expression of this protein completely protected the mice from demyelination. Interestingly, sCrry mice had low levels of demyelination at later times when control mice were remyelinating. Although the sCrry transgenic mice had lower levels of demyelination, there was no decrease in overall cellularity, however there were decreased numbers of microglia in the sCrry mice relative to controls. Strikingly, sCrry mice had early recovery of mature oligodendrocytes, although they later disappeared. TUNEL staining suggested that production of the sCrry protein in the transgenic mice protected from a late apoptosis event at 3 weeks of cuprizone treatment. Our data suggest complement provides some protection of mature oligodendrocytes during cuprizone treatment but may be critical for subsequent remyelination events. These data suggest that temporal restriction of complement inhibition may be required in some disease settings.

Characterization of the murine C3a receptor enhancer-promoter: expression control by an activator protein 1 sequence and an Ets-like site.

The complement anaphylatoxins, C3a and C5a, exert their effects by binding to their respective receptors. A number of studies have implicated these proteins in human disease, yet little is known about anaphylatoxin receptor gene regulation. In this report, we demonstrate that most of the regulatory functions in the murine C3aR gene lie within 50 bp of the transcription start site. This region is critical for macrophage expression but does not have activity in a non-expressing melanoma cell line. Within this small region are putative consensus binding sites for AP-1, NF-kappaB, Ets, and GATA transcription factors. Lack of a corresponding NF-kappaB site in the human sequence and lack of DNA binding activity in macrophage nuclear extracts suggests that the NF-kappaB site is nonfunctional. Luciferase data demonstrate that the GATA site functions as a negative regulatory element in RAW 264.7 macrophages. The AP-1 and Ets sites are critical for C3aR reporter gene expression, such that when each is mutated, a significant loss of activity is observed. Furthermore, we demonstrate that these sequences cooperate to mediate both basal and LPS-induced expression of C3aR. Interestingly, EMSA analyses demonstrate that the AP-1 site binds to c-Jun, and in vivo footprinting shows a typical footprint in this site, but the Ets site does not have a "typical" Ets footprint and does not bind to Ets-1/2 proteins in RAW 264.7 extracts. These data suggest that, although the control region for C3aR is small, interaction of several transcription factors can lead to complex patterns of gene regulation.

Expression of complement protein C5a in a murine mammary cancer model: tumor regression by interference with the cell cycle.

The C5a anaphylatoxin protein plays a central role in inflammation associated with complement activation. This protein is commonly regarded as one of the most potent inducers of the inflammatory response and a C5a peptide agonist was used as a molecular adjuvant. However, the full length C5a protein has not been tested as a potential tumor therapy. In this report, we describe the creation of a mini-gene construct that directs C5a expression to any cell of interest. Functional expression could be demonstrated in the murine mammary sarcoma, EMT6. When C5a expressing cells were injected into syngeneic mice, most C5a-expressing clones had significantly reduced tumor growth. Further characterization of a clone expressing low levels of C5a demonstrated that one-third of mice injected with this line had complete tumor regression. The mice whose tumors regressed were immune to subsequent challenge with unmodified EMT6 cells, suggesting that a component of the innate immune response can be used to augment adaptive immunity. Cellular analyses demonstrated that a significant difference in actual tumor cell number could be detected as early as day 10. A block in cell cycle progression was evident at all time points and high levels of apoptosis were observed early in the regression event. These data demonstrate that the complement protein C5a can play a significant protective role in tumor immunity.