Immunofocusing humoral immunity potentiates the functional efficacy of the AnAPN1 malaria transmission-blocking vaccine antigen.

Malaria transmission-blocking vaccines (TBVs) prevent the completion of the developmental lifecycle of malarial parasites within the mosquito vector, effectively blocking subsequent infections. The mosquito midgut protein Anopheline alanyl aminopeptidase N (AnAPN1) is the leading, mosquito-based TBV antigen. Structure-function studies identified two Class II epitopes that can induce potent transmission-blocking (T-B) antibodies, informing the design of the next-generation AnAPN1. Here, we functionally screened new immunogens and down-selected to the UF6b construct that has two glycine-linked copies of the T-B epitopes. We then established a process for manufacturing UF6b and evaluated in outbred female CD1 mice the immunogenicity of the preclinical product with the human-safe adjuvant Glucopyranosyl Lipid Adjuvant in a liposomal formulation with saponin QS21 (GLA-LSQ). UF6b:GLA-LSQ effectively immunofocused the humoral response to one of the key T-B epitopes resulting in potent T-B activity, underscoring UF6b as a prime TBV candidate to aid in malaria elimination and eradication efforts.

Development of a high-yield live-virus vaccine production platform using a novel fixed-bed bioreactor.

The increasing importance of viral vaccine manufacturing has driven the need for high cell density process optimization that allows for higher production levels. Vero cells are one of the more popular adherent cell lines used for viral vaccine production. However, production is limited due to the logistical limitations surrounding adherent cell line processes, such as large equipment footprints, time and labor-intensive processes, and larger costs per dose. We have addressed this limitation with the establishment of a viral vaccine production system utilizing the novel single use scale-X™ carbo bioreactor. The unit is compact and is scalable and one of the novel features of the system is the continuous in-line downstream purification and concentration processes associated with the bioreactor vessel. We present the results from a campaign featuring a proprietary Vero cell line for production of a live recombinant Vesicular stomatitis virus vaccine that features the Lassa Fever virus glycoproteins. Metabolite analyses and viral yield comparison between traditional flasks, cell factories, and the scale-X carbo bioreactor were performed, and on average, the single use bioreactor produced 2-4 logs higher titers per surface area, approximately 5 × 1010 pfu/cm2, compared to classical flatstock, 2.67 × 106 pfu/cm2, and cell factories production, 5.77 × 108 pfu/cm2. Overall, we describe a novel bioreactor platform that allows for a cost-efficient and scalable process for viral vaccine production.

Examination of vesicular stomatitis virus-induced morphology changes in individual Vero cells by QMod microscopy.

Viral infection of cultured cells induces changes in the biophysical characteristics of the affected cells. Advanced microscopic cameras such as Ovizio's QMod, coupled with the appropriate software, can measure a variety of characteristics on a per-cell basis. We have employed this system to monitor the progression of vesicular stomatitis virus infection in Vero cells and to describe the cellular changes associated with advancing vesicular stomatitis virus infection. The measurements of cellular characteristics are operator-independent, and the goal is to establish a robust method to mathematically determine viral infection levels in a given sample. This will provide a means to measure viral titer in a faster and less subjective way than manual reading of plaque assays or tissue culture infectious dose 50 assays.

Chikungunya virus infection in Cynomolgus macaques following Intradermal and aerosol exposure.

BACKGROUND: Chikungunya virus (CHIKV) is transmitted via mosquito bite and potentially by aerosol, causing chikungunya fever and arthritic disease in humans. There are currently no licensed vaccines or antiviral therapeutics to protect against CHIKV infection in humans. Animal models recapitulating human disease, especially for transmission by aerosol, are needed for licensure of such medical countermeasures. METHODS: Cynomolgus macaques (CMs) were challenged by intradermal (ID) inoculation or exposure to an aerosol containing CHIKV Ross strain at different target infectious doses (103-107 plaque forming units (PFU)). The clinical and virologic courses of disease were monitored up to 14 days post-exposure. RESULTS: ID infection of CMs led to overt clinical disease, detectable viremia, and increased blood markers of liver damage. Animals challenged by aerosol exhibited viremia and increased liver damage biomarkers with minimal observed clinical disease. All animals survived CHIKV challenge. CONCLUSIONS: We have described CHIKV infection in CMs following ID inoculation and, for the first time, infection by aerosol. Based on limited reported cases in the published literature, the aerosol model recapitulates the virologic findings of human infection via this route. The results of this study provide additional evidence for the potential use of CMs as a model for evaluating medical countermeasures against CHIKV.

Ectromelia Virus Disease Characterization in the BALB/c Mouse: A Surrogate Model for Assessment of Smallpox Medical Countermeasures.

In 2007, the United States- Food and Drug Administration (FDA) issued guidance concerning animal models for testing the efficacy of medical countermeasures against variola virus (VARV), the etiologic agent for smallpox. Ectromelia virus (ECTV) is naturally-occurring and responsible for severe mortality and morbidity as a result of mousepox disease in the murine model, displaying similarities to variola infection in humans. Due to the increased need of acceptable surrogate animal models for poxvirus disease, we have characterized ECTV infection in the BALB/c mouse. Mice were inoculated intranasally with a high lethal dose (125 PFU) of ECTV, resulting in complete mortality 10 days after infection. Decreases in weight and temperature from baseline were observed eight to nine days following infection. Viral titers via quantitative polymerase chain reaction (qPCR) and plaque assay were first observed in the blood at 4.5 days post-infection and in tissue (spleen and liver) at 3.5 days post-infection. Adverse clinical signs of disease were first observed four and five days post-infection, with severe signs occurring on day 7. Pathological changes consistent with ECTV infection were first observed five days after infection. Examination of data obtained from these parameters suggests the ECTV BALB/c model is suitable for potential use in medical countermeasures (MCMs) development and efficacy testing.

MicroRNA expression in mice infected with seasonal H1N1, swine H1N1 or highly pathogenic H5N1.

Influenza virus infections in humans remain a healthcare concern, and the need for vaccines, therapeutics and prophylactics remains a high priority. Understanding the molecular events associated with influenza-virus-induced pathology may lead to the identification of clinical disease biomarkers and novel antiviral targets. MicroRNAs (miRNAs) are well-conserved endogenous non-coding RNAs known to regulate post-transcriptional gene expression as well as play a major role in many biological processes and pathways. Animal studies have demonstrated that miRNAs are involved in viral disease and controlling inflammation. In this study, we examined the differences in the miRNA expression profiles associated with the lung in mice infected with influenza viruses that varied in virulence and pathogenicity. A statistical model was employed that utilized changes in miRNA expression to identify the virus that was used to infect the mice. This study identified a unique fingerprint of viral pathogenicity associated with seasonal H1N1, swine H1N1 and highly pathogenic H5N1 in the mouse model, and may lead to the identification of novel therapeutic and prophylactic targets.

Efficacy and safety of AVP-21D9, an anthrax monoclonal antibody, in animal models and humans.

Anthrax is an acute infectious disease caused by the spore-forming bacterium Bacillus anthracis. Timely administration of antibiotics approved for the treatment of anthrax disease may prevent associated morbidity and mortality. However, any delay in initiating antimicrobial therapy may result in increased mortality, as inhalational anthrax progresses rapidly to the toxemic phase of disease. An anthrax antitoxin, AVP-21D9, also known as Thravixa (fully human anthrax monoclonal antibody), is being developed as a therapeutic agent against anthrax toxemia. The efficacy of AVP-21D9 in B. anthracis-infected New Zealand White rabbits and in cynomolgus macaques was evaluated, and its safety and pharmacokinetics were assessed in healthy human volunteers. The estimated mean elimination half-life values of AVP-21D9 in surviving anthrax-challenged rabbits and nonhuman primates (NHPs) ranged from approximately 2 to 4 days and 6 to 11 days, respectively. In healthy humans, the mean elimination half-life was in the range of 20 to 27 days. Dose proportionality was observed for the maximum serum concentration (Cmax) of AVP-21D9 and the area under the concentration-time curve (AUC). In therapeutic efficacy animal models, treatment with AVP-21D9 resulted in survival of up to 92% of the rabbits and up to 67% of the macaques. Single infusions of AVP-21D9 were well tolerated in healthy adult volunteers across all doses evaluated, and no serious adverse events were reported. (This study has been registered at ClinicalTrials.gov under registration no. NCT01202695.).

Clinical profiles associated with influenza disease in the ferret model.

Influenza A viruses continue to pose a threat to human health; thus, various vaccines and prophylaxis continue to be developed. Testing of these products requires various animal models including mice, guinea pigs, and ferrets. However, because ferrets are naturally susceptible to infection with human influenza viruses and because the disease state resembles that of human influenza, these animals have been widely used as a model to study influenza virus pathogenesis. In this report, a statistical analysis was performed to evaluate data involving 269 ferrets infected with seasonal influenza, swine influenza, and highly pathogenic avian influenza (HPAI) from 16 different studies over a five year period. The aim of the analyses was to better qualify the ferret model by identifying relationships among important animal model parameters (endpoints) and variables of interest, which include survival, time-to-death, changes in body temperature and weight, and nasal wash samples containing virus, in addition to significant changes from baseline in selected hematology and clinical chemistry parameters. The results demonstrate that a disease clinical profile, consisting of various changes in the biological parameters tested, is associated with various influenza A infections in ferrets. Additionally, the analysis yielded correlates of protection associated with HPAI disease in ferrets. In all, the results from this study further validate the use of the ferret as a model to study influenza A pathology and to evaluate product efficacy.

Early indicators of disease in ferrets infected with a high dose of avian influenza H5N1.

Avian influenza viruses are widespread in birds, contagious in humans, and are categorized as low pathogenicity avian influenza or highly pathogenic avian influenza. Ferrets are susceptible to infection with avian and human influenza A and B viruses and have been widely used as a model to study pathogenicity and vaccine efficacy. In this report, the natural history of the H5N1 influenza virus A/Vietnam/1203/04 influenza infection in ferrets was examined to determine clinical and laboratory parameters that may indicate (1) the onset of disease and (2) survival. In all, twenty of 24 animals infected with 7 × 10(5) TCID(50) of A/Vietnam/1203/04 succumbed. A statistical analysis identified a combination of parameters including weight loss, nasal wash TCID(50), eosinophils, and liver enzymes such as alanine amino transferase that might possibly serve as indicators of both disease onset and challenge survival.

Animal models, prophylaxis, and therapeutics for arenavirus infections.

Arenaviruses are enveloped, bipartite negative single-stranded RNA viruses that can cause a wide spectrum of disease in humans and experimental animals including hemorrhagic fever. The majority of these viruses are rodent-borne and the arenavirus family can be divided into two groups: the Lassa-Lymphocytic choriomeningitis serocomplex and the Tacaribe serocomplex. Arenavirus-induced disease may include characteristic symptoms ranging from fever, malaise, body aches, petechiae, dehydration, hemorrhage, organ failure, shock, and in severe cases death. Currently, there are few prophylactic and therapeutic treatments available for arenavirus-induced symptoms. Supportive care and ribavirin remain the predominant strategies for treating most of the arenavirus-induced diseases. Therefore, efficacy testing of novel therapeutic and prophylactic strategies in relevant animal models is necessary. Because of the potential for person-to-person spread, the ability to cause lethal or debilitating disease in humans, limited treatment options, and potential as a bio-weapon, the development of prophylactics and therapeutics is essential. This article reviews the current arenavirus animal models and prophylactic and therapeutic strategies under development to treat arenavirus infection.

Implantation and monitoring of a novel telemetry unit in the Syrian golden hamster model.

Radiotelemetry allows for real-time remote monitoring of biological parameters in freely moving laboratory animals. The HD-X11 transmitter is a novel telemetry device that enables simultaneous collection of body temperature, activity, blood pressure, electrocardiogram (ECG), and other biopotentials in small animal models. Previously, researchers could only collect either blood pressure or ECG parameters; prioritizing the signal of most interest or increasing the number of animals on study to capture both signals at one time. This new device eliminates the need for separate animal groups for assorted measurements and allows for a more complete cardiovascular assessment. Evaluation of the transmitter from both surgical and data collection perspectives indicates that the HD-X11 transmitter can be a useful tool to researchers in a wide range of scientific and medical fields.

Efficacy of a heterologous vaccine and adjuvant in ferrets challenged with influenza virus H5N1.

BACKGROUND: In 1997, highly pathogenic avian influenza (HPAI) viruses caused outbreaks of disease in domestic poultry markets in Hong Kong. The virus has also been detected in infected poultry in Europe and Africa. OBJECTIVE: The objective of this study was to determine the efficacy of a heterologous vaccine administered with and without the aluminum hydroxide adjuvant in ferrets challenged with HPAI (A/Vietnam/1203/04). METHODS: Animals in four of the five groups were vaccinated twice 21 days apart, with two doses of a heterologous monovalent subvirion vaccine with or without an aluminum hydroxide adjuvant and challenged with a lethal target dose of A/Vietnam/1203/04. RESULTS: All animals vaccinated with the heterologous vaccine in combination with the aluminum hydroxide adjuvant survived a lethal challenge of A/Vietnam/1203/04. Four of the eight animals vaccinated with 30 µg of the vaccine without the adjuvant survived, while two of the eight animals vaccinated with 15 µg of the vaccine without the adjuvant survived. None of the unvaccinated control animals survived challenge. Additionally, changes in virus recovered from nasal washes and post-mortem tissues and serology suggest vaccine efficacy. CONCLUSIONS: Altogether, the data suggest that the heterologous vaccine in combination with the aluminum hydroxide adjuvant offers maximum protection against challenge with A/Vietnam/1203/04 when compared to the unvaccinated control animals or animals vaccinated without any adjuvant.

Inhibition of Lassa virus and Ebola virus infection in host cells treated with the kinase inhibitors genistein and tyrphostin.

Arenaviruses and filoviruses are capable of causing hemorrhagic fever syndrome in humans. Limited therapeutic and/or prophylactic options are available for humans suffering from viral hemorrhagic fever. In this report, we demonstrate that pre-treatment of host cells with the kinase inhibitors genistein and tyrphostin AG1478 leads to inhibition of infection or transduction in cells infected with Ebola virus, Marburg virus, and Lassa virus. In all, the results demonstrate that a kinase inhibitor cocktail consisting of genistein and tyrphostin AG1478 is a broad-spectrum antiviral that may be used as a therapeutic or prophylactic against arenavirus and filovirus hemorrhagic fever.

Attenuation of neurovirulence, biodistribution, and shedding of a poliovirus:rhinovirus chimera after intrathalamic inoculation in Macaca fascicularis.

A dependence of poliovirus on an unorthodox translation initiation mode can be targeted selectively to drive viral protein synthesis and cytotoxicity in malignant cells. Transformed cells are naturally susceptible to poliovirus, due to widespread ectopic upregulation of the poliovirus receptor, Necl-5, in ectodermal/neuroectodermal cancers. Viral tumor cell killing and the host immunologic response it engenders produce potent, lasting antineoplastic effects in animal tumor models. Clinical application of this principle depends on unequivocal demonstration of safety in primate models for paralytic poliomyelitis. We conducted extensive dose-range-finding, toxicity, biodistribution, shedding, and neutralizing antibody studies of the prototype oncolytic poliovirus recombinant, PVS-RIPO, after intrathalamic inoculation in Macaca fascicularis. These studies suggest that intracerebral PVS-RIPO inoculation does not lead to viral propagation in the central nervous system (CNS), does not cause histopathological CNS lesions or neurological symptoms that can be attributed to the virus, is not associated with extraneural virus dissemination or replication and does not induce shedding of virus with stool. Intrathalamic PVS-RIPO inoculation induced neutralizing antibody responses against poliovirus serotype 1 in all animals studied.

Differences in craniofacial and dental characteristics of adolescent Mexican Americans and European Americans.

INTRODUCTION: The purpose of this study was to compare the soft-tissue profiles of matched Class I adolescent European Americans and Mexican Americans. The secondary aim was to explain profile differences based on group differences in soft-tissue thickness, skeletal morphology, dental position, and tooth size. METHODS: The study pertained to 207 untreated Class I adolescents, including 93 Mexican Americans and 114 European Americans. Lateral cephalometric and model analyses were performed to quantify morphologic differences. Two-way analyses of variance were used to evaluate ethnicity, sex, and their interaction. RESULTS: Mexican Americans had significantly (P <0.05) greater lip protrusion and facial convexity than did European Americans. Mexican Americans had smaller craniofacial dimensions and larger teeth, resulting in maxillary and mandibular dentoalveolar protrusion. Mexican Americans also had thicker soft tissues and greater maxillary skeletal prognathism than European Americans. The combination of thicker soft tissues, maxillary skeletal prognathism, and dentoalveolar protrusion explained the protrusive lips of Mexican Americans. The greater facial convexity of Mexican Americans was due primarily to maxillary prognathism and mandibular hyperdivergence. Sex differences pertained primarily to size; the linear dimensions of the boys were consistently and significantly larger than those of the girls. CONCLUSIONS: European American normative data and treatment objectives do not apply to Mexican Americans. Knowledge of the soft-tissue, skeletal morphology, and dental position differences should be applied when planning treatment for Mexican American patients.

Genistein, a general kinase inhibitor, as a potential antiviral for arenaviral hemorrhagic fever as described in the Pirital virus-Syrian golden hamster model.

Arenaviruses are rodent-borne negative strand RNA viruses and infection of these viruses in humans may result in disease and hemorrhagic fever. To date, supportive care, ribavirin, and in some cases immune plasma remain the foremost treatment options for arenaviral hemorrhagic fever. Research with the hemorrhagic fever causing-arenaviruses usually requires a Biosafety level (BSL)-4 environment; however, surrogate animal model systems have been developed to preliminarily study and screen various vaccines and antivirals. The Syrian golden hamster-Pirital virus (PIRV) surrogate model of hemorrhagic fever provides an opportunity to test new antivirals in an ABSL-3 setting. Thus, we challenged hamsters, implanted with telemetry, with PIRV and observed viremia and tissue viral titers, and changes in core body temperature, hematology, clinical chemistry, and coagulation parameters. Physical signs of disease of the PIRV-infected hamsters included weight loss, lethargy, petechial rashes, epistaxis, ocular orbital and rectal hemorrhage, and visible signs of neurologic disorders. However, treating animals with genistein, a plant derived isoflavone and general kinase inhibitor, resulted in increased survival rates and led to an improved clinical profile. In all, the results from this study demonstrate the potential of a general kinase inhibitor genistein as an antiviral against arenaviral hemorrhagic fever.

Developmental regulation of Neuregulin1 isoforms and erbB receptor expression in intact rat dorsal root ganglia.

Neuregulins (NRGs) are a family of growth factors which bind to the erbB family of tyrosine kinase receptors. The exact nature and interaction of specific NRG isoforms and erbB receptors that occur during the development of the nervous system have not been reported. In order to better understand the role that different NRG isoforms and erbB receptors play in the differentiation, proliferation, and survival of neurons and glial cells, we isolated protein and mRNA from dorsal root ganglia of rat pups between embryonic day (E) 13 and postnatal day (P) 15. The relative expression levels of the NRGs and erbB receptors for the different time points were compared using both Western and RT-PCR analyses. NRG1-type1alpha protein levels were highest at E-13 and then decreased by approximately 40% and remained constant through P-15. In contrast, mRNA levels for NRG1-type1alpha remained constant from E-15 to P-15. The protein levels for NRG1-type 1beta were similar to NRG1-type1alpha at E-13 with an approximate 40% increase in the levels at E-15 and E-17 followed by a decrease to E-13 levels for the remainder of the developmental time periods. The mRNA levels for NRG1-type1beta remained constant from E-15 to P-15. The protein and mRNA expression patterns for each erbB receptor were distinctive. The protein levels for erbB-2 were highest at E-19 while erbB-3 levels were highest at E-17 and E-18. ErbB-4 protein levels were highest at E-13 and decreased through P-15. The developmental pattern for erbB-2 and erbB-4 mRNA levels had no relation to that of the corresponding protein levels while the mRNA levels for erbB-3 were highest at E-17 and E-18 similar to the pattern observed for the erbB-3 protein levels. We concluded that both NRG and erbB expression in dorsal root ganglia are mostly translationally controlled and that NRG1 isoforms and their erbB receptors are not coordinately regulated.

Pichindé virus is trafficked through a dynamin 2 endocytic pathway that is dependent on cellular Rab5- and Rab7-mediated endosomes.

Pichindé virus (PICV) is a New World arenavirus that has been shown to enter cells through a clathrin-dependent endocytic pathway. In this study, we determined that PICV is trafficked through the cellular dynamin 2 (dyn2) endocytic pathway. Additionally, the data suggest that PICV entry is pH-dependent and that the virus travels through Rab5-mediated early and Rab7-mediated late endosomes. In all, this study characterizes the endocytic pathway utilized by the arenavirus PICV.

Exploring kinase inhibitors as therapies for human arenavirus infections.

Arenaviruses are rodent-borne RNA viruses, and some have the capacity to cause hemorrhagic fever and death in infected individuals and thus have been identified as a potential bioterrorism threat. Ribavirin and supportive care are currently the approved therapeutic options for individuals suffering from arenavirus-induced hemorrhagic fever. However, new research has suggested that immune plasma treatment or kinase inhibitors may provide a therapeutic option for treating arenavirus infections in humans. This article puts forth a perspective as to the potential use of kinase inhibitors as an antiviral therapeutic for arenavirus infections.

Genistein treatment of cells inhibits arenavirus infection.

Arenaviridae is a family of enveloped viruses some of which are capable of causing hemorrhagic fever syndromes in humans. In this report, we demonstrate that treatment of host cells with the tyrosine kinase inhibitor genistein inhibits infection of cells with the New World arenavirus Pichindé (PICV). The greatest degree of inhibition was observed in pre-treated target cells, but modest inhibition of infection was also seen when drug was added to cultures up to 48h after infection. We show that PICV-induced phosphorylation of the activating transcription factor-2 protein (ATF-2) and cyclic adenosine monophosphate response element binding protein (CREB) is inhibited following genistein treatment. Lastly, genistein treatment also inhibited transduction of cells with pseudotyped retrovirus particles expressing envelope proteins of the Old World arenavirus Lassa virus. These results demonstrate that kinase activity is required for arenavirus infection and that therapeutics designed to inhibit kinase activity should be explored.