Harry Davis

Safety, immunogenicity, and efficacy of the ML29 reassortant vaccine for Lassa fever in small non-human primates

A single injection of ML29 reassortant vaccine for Lassa fever induces low, transient viremia, and low or moderate levels of ML29 replication in tissues of common marmosets depending on the dose of the vaccination. The vaccination elicits specific immune responses and completely protects marmosets against fatal disease by induction of sterilizing cell-mediated immunity. DNA array analysis of human peripheral blood mononuclear cells from healthy donors exposed to ML29 revealed that gene expression patterns in ML29-exposed PBMC and control, media-exposed PBMC, clustered together confirming safety profile of the ML29 in non-human primates. The ML29 reassortant is a promising vaccine candidate for Lassa fever.

Early Blood Profiles of Virus Infection in a Monkey Model for Lassa Fever

ABSTRACT Acute arenavirus disease in primates, like Lassa hemorrhagic fever in humans, begins with flu-like symptoms and leads to death approximately 2 weeks after infection. Our goal was to identify molecular changes in blood that are related to disease progression. Rhesus macaques (Macaca mulatta) infected intravenously with a lethal dose of lymphocytic choriomeningitis virus (LCMV) provide a model for Lassa virus infection of humans. Blood samples taken before and during the course of infection were used to monitor gene expression changes that paralleled disease onset. Changes in blood showed major disruptions in eicosanoid, immune response, and hormone response pathways. Approximately 12% of host genes alter their expression after LCMV infection, and a subset of these genes can discriminate between virulent and nonvirulent LCMV infection. Major transcription changes have been given preliminary confirmation by quantitative PCR and protein studies and will be valuable candidates for future validation as biomarkers for arenavirus disease.

Immune activation, viral gene product expression and neurotoxicity in the HIV-1 transgenic rat

The HIV-1 transgenic (TG) rat has been shown to be a useful model of nervous system disease that occurs in human HIV-1 infection. Studies were, therefore, performed to examine characteristics of the immune response in the periphery and brain of the animals and expression of factors in the nervous system that might be associated with neurotoxicity. Activated splenocytes from wild-type (WT) and TG rats were stimulated with either CD3/CD28 or with lipopolysaccharide (LPS) and examined for proliferative responses and for proinflammatory cytokine (IFN-γ, TNF-α and IL-1β) secretion. Brain tissue lysates from the rats were also examined for proinflammatory cytokine levels and tissue sections were stained by immunofluorescence for class II MHC+, ED1+ or Iba1+ (for macrophages and microglial cells), and for GFAP+ (for astrocytes) cells and for co-labeling of these cells for TNF-α. Co-labeling was also performed to identify cells expressing HIV-1 gp160, tat, nef and vif. Finally, on Western blots brain tissue lysates were examined for phosphorylation of Erk1/2, p38, JNK-SAPK and Erk5. TG rat splenocyte proliferative responses were higher than for WT with CD3/CD28-stimulation but lower than WT with LPS stimulation. CD3/CD28-stimulated TG rat splenocytes also secreted higher levels of IFN-γ, TNF-α and IL-1β whereas LPS-stimulated TG rat splenocytes secreted higher levels of only TNF-α than cultures from WT rats. Levels of all three cytokines were higher in brain lysates from TG rats than for WT rats. On immunofluorescence staining of corresponding sections of brain, TG rats contained increased numbers of class II MHC+ and ED1+ cells, and there was also increased co-labeling or these cells as well as astrocytes for TNF-α. Iba1+ cells showed positive staining for all of the HIV proteins whereas astrocytes showed significant positive staining for only nef and vif. Phosphorylation of Erk1/2, p38 and JNK/SAPK was detected for both TG and WT rat tissues with higher levels of phosphorylation forms of these proteins detected in the TG rat brain. Phosphorylation of Erk5, a marker that is associated with specifically neuronal repair, was detected only in TG rat brain. These studies suggest that activated nervous system mononuclear phagocytes and astrocytes expressing HIV-1 gene products in specific patterns are associated with neurodegeneration in the HIV-1 TG rat.

Gene expression in primate liver during viral hemorrhagic fever

BackgroundRhesus macaques infected with lymphocytic choriomeningitis virus (LCMV) provide a model for human Lassa fever. Disease begins with flu-like symptoms and progresses rapidly with fatal consequences. Previously, we profiled the blood transcriptome of LCMV-infected monkeys (M. Djavani et al J. Virol. 2007) showing distinct pre-viremic and viremic stages that discriminated virulent from benign infections. In the present study, changes in liver gene expression from macaques infected with virulent LCMV-WE were compared to gene expression in uninfected monkeys as well as to monkeys that were infected but not diseased.ResultsBased on a functional pathway analysis of differentially expressed genes, virulent LCMV-WE had a broader effect on liver cell function than did infection with non-virulent LCMV-Armstrong. During the first few days after infection, LCMV altered expression of genes associated with energy production, including fatty acid and glucose metabolism. The transcriptome profile resembled that of an organism in starvation: mRNA for acetyl-CoA carboxylase, a key enzyme of fatty acid synthesis was reduced while genes for enzymes in gluconeogenesis were up-regulated. Expression was also altered for genes associated with complement and coagulation cascades, and with signaling pathways involving STAT1 and TGF-β.ConclusionMost of the 4500 differentially expressed transcripts represented a general response to both virulent and mild infections. However, approximately 250 of these transcripts had significantly different expression in virulent infections as compared to mild infections, with approximately 30 of these being differentially regulated during the pre-viremic stage of infection. The genes that are expressed early and differently in mild and virulent disease are potential biomarkers for prognosis and triage of acute viral disease.

An attenuated Lassa vaccine in SIV-infected rhesus macaques does not persist or cause arenavirus disease but does elicit Lassa virus-specific immunity

BackgroundLassa hemorrhagic fever (LHF) is a rodent-borne viral disease that can be fatal for human beings. In this study, an attenuated Lassa vaccine candidate, ML29, was tested in SIV-infected rhesus macaques for its ability to elicit immune responses without instigating signs pathognomonic for arenavirus disease. ML29 is a reassortant between Lassa and Mopeia viruses that causes a transient infection in non-human primates and confers sterilizing protection from lethal Lassa viral challenge. However, since the LHF endemic area of West Africa also has high HIV seroprevalence, it is important to determine whether vaccination could be safe in the context of HIV infection.ResultsSIV-infected and uninfected rhesus macaques were vaccinated with the ML29 virus and monitored for specific humoral and cellular immune responses, as well as for classical and non-classical signs of arenavirus disease. Classical disease signs included viremia, rash, respiratory distress, malaise, high liver enzyme levels, and virus invasion of the central nervous system. Non-classical signs, derived from profiling the blood transcriptome of virulent and non-virulent arenavirus infections, included increased expression of interferon-stimulated genes (ISG) and decreased expression of COX2, IL-1β, coagulation intermediates and nuclear receptors needed for stress signaling. All vaccinated monkeys showed ML29-specific antibody responses and ML29-specific cell-mediated immunity.ConclusionSIV-infected and uninfected rhesus macaques responded similarly to ML29 vaccination, and none developed chronic arenavirus infection. Importantly, none of the macaques developed signs, classical or non-classical, of arenavirus disease.

Toxicity, antimicrobial and anthelmintic activities of Vernonia guineensis Benth. (Asteraceae) crude extracts

ETHNOPHARMACOLOGICAL RELEVANCE This study examined the antibacterial, antifungal, and anthelmintic properties of extracts obtained from the plant Vernonia guineensis, a plant commonly used in traditional Cameroonian medicine. MATERIALS AND METHODS For in vitro studies, 10 g of leaf and tuber powder from V. guineensis was extracted separately using dichloromethane, methanol and distilled water. The extracts were dried in vacuo and used for antimicrobial and anthelmintic activity studies. In the antimicrobial assay, extracts were tested against bacterial and fungal organisms including; Staphylococcus aureus, Staphylococcus epidermidis, Acinetobacter baumannii, Aspergillus fumigatus, Candida albicans and Trichophyton mentagrophytes. In the anthelmintic assay, larval and adult stages of the hookworm Ancylostoma ceylanicum and the mouse nematode Trichuris muris were used. For the acute toxicity test, male and female rats of 150-200 g body weight were used in the experiment. The aqueous extract of V. guineensis tubers was administered in 4 doses of 500, 1000, 2000 and 4000 mg/kg per group (n=6), respectively, and the control group received distilled water. RESULTS The crude extracts exhibited weak antibacterial and antifungal activity except for the dichloromethane extract, which showed moderate activity against A. fumigatus (MIC=200 μg/ml). In the anthelmintic assay, the organic extracts of the tubers had 100% killing efficacy against T. muris at 2mg/ml in 48 h, while the aqueous extract showed no activity. The organic leaf extracts demonstrated potent activity killing 100% of the adult worms 1mg/ml in 24h. The aqueous leaf extract was active at 2mg/ml in 72 h, killing 100% of the adult worms. In the acute toxicity test, V. guineensis did not produce any toxic signs or death at the maximum concentration of 4000 mg/kg. CONCLUSION Crude extracts from V. guineensis possess anthelmintic activity against T. muris with only weak antibiotic activity. Acute administration of aqueous extract from V. guineensis tubers did not produce toxic effects in rats. The absence of acute toxicity at the highest concentration tested indicates that the tea decoction from V. guineensis extract is safe at concentrations ≤ 4000 mg/kg.

Cytotoxic sesquiterpene lactones from the leaves of Vernonia guineensis Benth. (Asteraceae)

ETHNOPHARMACOLOGICAL RELEVANCE Vernonia guineensis Benth. (Asteraceae) preparations are used in folk medicine in Cameroon to treat a number of ailments, including prostate cancer and malaria, and is used as an anthelmintic, adaptogen and antidote. The aim of this study was to continue the validation of the activity of Vernonia guineensis Benth. extracts and isolated molecules against cancer cell lines following the previous isolation of an anti-prostate cancer sugar ester from the root extract. MATERIALS AND METHODS Acetone extracts of Vernonia guineensis Benth. leaves were tested for activity against 10 cancer cell lines (Breast-MDA-MB-231, Breast-MCF-7, Colon-HCT-116, Leukemia-HL-60, Lung-A549, Melanoma-A375, Ovarian-OVCAR3, Pancreas-Mia-paca, Prostate-PC-3 and Prostate-DU-145). The acetone extract was subjected to bioactivity guided fractionation. Anti-proliferation and clonogenic activity of the isolated compounds were tested. The WST-1 assay was used for the anti-proliferation activity, while the standard clonogenic test was used to determine the clonogenic activity. RESULTS The acetone extract of Vernonia guineensis Benth. demonstrated in vitro activity ranging from IC50 4-26μg/mL against the 10 cell lines. Activity guided fractionation of this extract yielded two sesquiterpene lactones, isolated for the first time from the genus Vernonia. The compounds were characterized using spectroscopic experiments, including a combination of 1D and 2D NMR data. Vernopicrin (1) and Vernomelitensin (2) demonstrated in vitro activity against human cancer cell lines with IC50 ranging from 0.35-2.04μM (P<0.05) and 0.13-1.5μM (P<0.05), respectively, between the most and least sensitive cell lines for each compound. Vernopicrin was most active against the human melanoma (A375) cell line and least active against the lung cancer (A549) cell line, while Vernomelitensin was also most active against the human melanoma (A375) cell line and least active against the breast cancer (MCF-7) cell line. Both compounds also demonstrated anticlonogenic activity. CONCLUSION The cytotoxicity demonstrated by the crude extract and isolated sesquiterpenes against cancer cell lines highlights the medicinal potential of V. guineensis. The selective anti-proliferation and dose dependent anticlonogenic activities suggest that the identified sesquiterpenes could be potential antitumor agents.

Evaluation of Lassa virus vaccine immunogenicity in a CBA/J-ML29 mouse model

Lassa fever (LF) is one of the most prevalent viral hemorrhagic fevers in West Africa responsible for thousands of deaths annually. The BSL-4 containment requirement and lack of small animal model to evaluate Lassa virus (LASV)-specific cell-mediated immunity (CMI) complicate development of effective LF vaccines. Here we have described a CBA/J-ML29 model allowing evaluation of LASV-specific CMI responses in mice. This model is based on Mopeia virus reassortant clone ML29, an attractive immunogenic surrogate for LASV. A single intraperitoneal (i.p.) immunization of CBA/J mice with ML29 protected animals against a lethal homologous intracerebral (i.c.) challenge with 588 LD(50). The ML29-immunized mice displayed negligible levels of LASV-specific antibody titers, but LASV-specific CMI responses were detectable early and peaked on day 8-10 after immunization. A T cell cytotoxicity assay in vivo showed a correlation between LASV-specific cytotoxicity and the timing of protection induced by the ML29 immunization. Notably, CBA/J mice that received CD8+ T cell-depleted splenocytes from ML29-immunized donors all succumbed to a lethal i.c. challenge, demonstrating that CD8+ T cells are critical in protection. The CBA/J-ML29 model can be useful immunological tool for the preliminary evaluation of immunogenicity and efficacy of vaccine candidates against LASV outside of BSL-4 containment facilities.

In vitro anti-prostate cancer and ex vivo antiangiogenic activity of Vernonia guineensis Benth. (Asteraceae) tuber extracts

ETHNOPHARMACOLOGICAL RELEVANCE Prostate cancer is a major problem worldwide and affects most men above the age of forty-five. Vernonia guineensis Benth. (Asteraceae) root decoction is used in folk medicine in Cameroon to treat a number of ailments including prostate cancer. The aim of this study was to provide a preliminary validation of the use of Vernonia guineensis Benth. extracts to treat prostate cancer by evaluating the in vitro activity of its crude extracts and isolated molecules on prostate cancer cells lines and effect on angiogenesis which is essential for growth and metastases of prostate cancer. MATERIALS AND METHODS Aqueous, dichloromethane and methanol extracts of Vernonia guineensis Benth. tubers were tested for activity against three prostate cancer cell lines (PC-3, DU-145 and AT3B-1). The dichloromethane extract was subjected to bioactivity guided fractionation. Anti-proliferation, clonogenic and antiangiogenic activity of the crude extracts and isolated compound were tested. The WST-1 assay was used for the anti-proliferation activity meanwhile the standard clonogenic test and the rat ring aorta assay were carried out to determine the clonogenic and antiangiogenic activity of tested products respectively. RESULTS The aqueous and methanol extracts of Vernonia guineensis Benth. demonstrated weak activity against prostate cancer cell lines in vitro with IC(50)>100 μg/mL. The dichloromethane extract was more potent with IC(50) of 56.233±3.630 μg/ml and 67.316±2.452 μg/ml against the DU-145 and PC-3 cell lines respectively. Activity guided fractionation of this extract yielded a Pentaisovalerylsucrose (1) isolated for the first time from a natural source to the best of our knowledge. Compound 1 demonstrated in vitro activity against the human prostate cancer cell lines PC-3 and DU-145 with IC(50) of 5.701±0.142 μM and 4.275±0.710 μM, respectively. The IC(50) of the compound was 5.763±0.425 μM against AT3B-1, a rat prostate cancer cell line expressing P-glycoprotein which is linked to drug resistance in most metastatic cancers. Compared to compound 1, Paclitaxel and Docetaxel were active against AT3B-1 at 2.641±1.253 μM and 0.613±0.251 μM. Paclitaxel showed IC(50) values of 0.004±0.002 μM and 0.003±0.001 μM against DU-145 and PC-3 prostate cancer cell lines respectively. Docetaxel showed IC(50) values of 0.002±0.001 μM and 0.004±0.001 μM against DU-145 and PC-3 prostate cancer cell lines respectively. CONCLUSION The in vitro anti-prostate cancer and the antiangiogenic activity of Vernonia guineensis Benth. extracts and isolated compound support the use of the tubers of this plant for the treatment of prostate cancer.

In vivo antiprostate tumor potential of Vernonia guineensis Benth. (Asteraceae) tuber extract (VGDE) and the cytotoxicity of its major compound pentaisovaleryl sucrose.

ETHNOPHARMACOLOGICAL RELEVANCE Vernonia guineensis Benth. (Asteraceae) root decoction is used in folk medicine in Cameroon to treat some ailments including prostate cancer. The aim of this study was to validate the claimed antiprostate cancer activity of Vernonia guineensis Benth. in vivo and to investigate the cytotoxicity of a pentaisovaleryl sucrose isolated from Vernonia guineensis on some cancer cell lines. MATERIALS AND METHODS A crude dichloromethane extract of Vernonia guineensis (VGDE) was used for this study. For in vivo antiprostate cancer efficacy, nude mice (n=16) were injected subcutaneously with prostate cancer PC-3 cells. Upon the formation of the xenograft tumors, the mice were divided into two equal groups with approximately the same mean tumor volume per group. One group was treated with VGDE orally (500 mg/kg) and the other with a vehicle control for 30 days. Body weight and tumor volumes were measured 2× a week and on the 33rd day, the mice were euthanized and tumors harvested and weighed. For the cytotoxicity study, the WST-1 assay was used to determine the activity of pentaisovaleryl sucrose previously isolated from VGDE. The cancer cell lines used in the cytotoxicity study included breast, colon, leukemia, lung, melanoma, ovarian and prostate. RESULTS Prostate cancer (PC-3) xenograft tumors treated with VGDE showed a significant decrease in tumor size (P=0.0295) compared to control. Pentaisovaleryl sucrose also demonstrated cytotoxicity against various cancer cell lines with IC₅₀ values as follows: MDA-MD-231-6.66µM; MCF-7-7.50 µM; HCT116-14.12 µM; A549-5.76 µM; HL60-6.43 µM; A375-8.64 µM; OVCAR3-9.53 µM; Capan1-7.13 µM; Mia-Paca 6.47 µM. CONCLUSION VGDE does possess in vivo activity against prostate tumor and has potential for development into a natural product for the treatment of prostate cancer. This study thus provides preliminary validation for the folk use of Vernonia guineensis against prostate conditions. Further in vivo studies are however required to confirm these results and to understand the mechanism of action of VGDE and the in vivo efficacy of pentaisovaleryl sucrose.