[18F]-Fluorodeoxyglucose Uptake in Lymphoid Tissue Serves as a Predictor of Disease Outcome in the Nonhuman Primate Model of Monkeypox Virus Infection.

Real-time bioimaging of infectious disease processes may aid countermeasure development and lead to an improved understanding of pathogenesis. However, few studies have identified biomarkers for monitoring infections using in vivo imaging. Previously, we demonstrated that positron emission tomography/computed tomography (PET/CT) imaging with [18F]-fluorodeoxyglucose (FDG) can monitor monkeypox disease progression in vivo in nonhuman primates (NHPs). In this study, we investigated [18F]-FDG-PET/CT imaging of immune processes in lymphoid tissues to identify patterns of inflammation in the monkepox NHP model and to determine the value of [18F]-FDG-PET/CT as a biomarker for disease and treatment outcomes. Quantitative analysis of [18F]-FDG-PET/CT images revealed differences between moribund and surviving animals at two sites vital to the immune response to viral infections, bone marrow and lymph nodes (LNs). Moribund NHPs demonstrated increased [18F]-FDG uptake in bone marrow 4 days postinfection compared to surviving NHPs. In surviving, treated NHPs, increase in LN volume correlated with [18F]-FDG uptake and peaked 10 days postinfection, while minimal lymphadenopathy and higher glycolytic activity were observed in moribund NHPs early in infection. Imaging data were supported by standard virology, pathology, and immunology findings. Even with the limited number of subjects, imaging was able to differentiate the difference between disease outcomes, warranting additional studies to demonstrate whether [18F]-FDG-PET/CT can identify other, subtler effects. Visualizing altered metabolic activity at sites involved in the immune response by [18F]-FDG-PET/CT imaging is a powerful tool for identifying key disease-specific time points and locations that are most relevant for pathogenesis and treatment.IMPORTANCE Positron emission tomography and computed tomography (PET/CT) imaging is a universal tool in oncology and neuroscience. The application of this technology to infectious diseases is far less developed. We used PET/CT imaging with [18F]-labeled fluorodeoxyglucose ([18F]-FDG) in monkeys after monkeypox virus exposure to monitor the immune response in lymphoid tissues. In lymph nodes of surviving monkeys, changes in [18F]-FDG uptake positively correlated with enlargement of the lymph nodes and peaked on day 10 postinfection. In contrast, the bone marrow and lymph nodes of nonsurvivors showed increased [18F]-FDG uptake by day 4 postinfection with minimal lymph node enlargement, indicating that elevated cell metabolic activity early after infection is predictive of disease outcome. [18F]-FDG-PET/CT imaging can provide real-time snapshots of metabolic activity changes in response to viral infections and identify key time points and locations most relevant for monitoring the development of pathogenesis and for potential treatment to be effective.

Exposure of rhesus monkeys to cowpox virus Brighton Red by large-particle aerosol droplets results in an upper respiratory tract disease.

We previously demonstrated that small-particle (0.5-3.0 µm) aerosol infection of rhesus monkeys (Macaca mulatta) with cowpox virus (CPXV)-Brighton Red (BR) results in fulminant respiratory tract disease characterized by severe lung parenchymal pathology but only limited systemic virus dissemination and limited classic epidermal pox-like lesion development (Johnson et al., 2015). Based on these results, and to further develop CPXV as an improved model of human smallpox, we evaluated a novel large-particle aerosol (7.0-9.0 µm) exposure of rhesus monkeys to CPXV-BR and monitored for respiratory tract disease by serial computed tomography (CT). As expected, the upper respiratory tract and large airways were the major sites of virus-induced pathology following large-particle aerosol exposure. Large-particle aerosol CPXV exposure of rhesus macaques resulted in severe upper airway and large airway pathology with limited systemic dissemination.

Preclinical Development of Inactivated Rabies Virus-Based Polyvalent Vaccine Against Rabies and Filoviruses.

We previously described the generation of a novel Ebola virus (EBOV) vaccine based on inactivated rabies virus (RABV) containing EBOV glycoprotein (GP) incorporated in the RABV virion. Our results demonstrated safety, immunogenicity, and protective efficacy in mice and nonhuman primates (NHPs). Protection against viral challenge depended largely on the quality of the humoral immune response against EBOV GP.Here we present the extension and improvement of this vaccine by increasing the amount of GP incorporation into virions via GP codon-optimization as well as the addition of Sudan virus (SUDV) and Marburg virus (MARV) GP containing virions. Immunogenicity studies in mice indicate similar immune responses for both SUDV GP and MARV GP compared to EBOV GP. Immunizing mice with multiple antigens resulted in immune responses similar to immunization with a single antigen. Moreover, immunization of NHP with the new inactivated RABV EBOV vaccine resulted in high titer neutralizing antibody levels and 100% protection against lethal EBOV challenge when applied with adjuvant.Our results indicate that an inactivated polyvalent vaccine against RABV filoviruses is achievable. Finally, the novel vaccines are produced on approved VERO cells and a clinical grade RABV/EBOV vaccine for human trials has been produced.

Ebola virus modulates transforming growth factor ß signaling and cellular markers of mesenchyme-like transition in hepatocytes.

UNLABELLED: Ebola virus (EBOV) causes a severe hemorrhagic disease in humans and nonhuman primates, with a median case fatality rate of 78.4%. Although EBOV is considered a public health concern, there is a relative paucity of information regarding the modulation of the functional host response during infection. We employed temporal kinome analysis to investigate the relative early, intermediate, and late host kinome responses to EBOV infection in human hepatocytes. Pathway overrepresentation analysis and functional network analysis of kinome data revealed that transforming growth factor (TGF-ß)-mediated signaling responses were temporally modulated in response to EBOV infection. Upregulation of TGF-ß signaling in the kinome data sets correlated with the upregulation of TGF-ß secretion from EBOV-infected cells. Kinase inhibitors targeting TGF-ß signaling, or additional cell receptors and downstream signaling pathway intermediates identified from our kinome analysis, also inhibited EBOV replication. Further, the inhibition of select cell signaling intermediates identified from our kinome analysis provided partial protection in a lethal model of EBOV infection. To gain perspective on the cellular consequence of TGF-ß signaling modulation during EBOV infection, we assessed cellular markers associated with upregulation of TGF-ß signaling. We observed upregulation of matrix metalloproteinase 9, N-cadherin, and fibronectin expression with concomitant reductions in the expression of E-cadherin and claudin-1, responses that are standard characteristics of an epithelium-to-mesenchyme-like transition. Additionally, we identified phosphorylation events downstream of TGF-ß that may contribute to this process. From these observations, we propose a model for a broader role of TGF-ß-mediated signaling responses in the pathogenesis of Ebola virus disease. IMPORTANCE: Ebola virus (EBOV), formerly Zaire ebolavirus, causes a severe hemorrhagic disease in humans and nonhuman primates and is the most lethal Ebola virus species, with case fatality rates of up to 90%. Although EBOV is considered a worldwide concern, many questions remain regarding EBOV molecular pathogenesis. As it is appreciated that many cellular processes are regulated through kinase-mediated phosphorylation events, we employed temporal kinome analysis to investigate the functional responses of human hepatocytes to EBOV infection. Administration of kinase inhibitors targeting signaling pathway intermediates identified in our kinome analysis inhibited viral replication in vitro and reduced EBOV pathogenesis in vivo. Further analysis of our data also demonstrated that EBOV infection modulated TGF-ß-mediated signaling responses and promoted "mesenchyme-like" phenotypic changes. Taken together, these results demonstrated that EBOV infection specifically modulates TGF-ß-mediated signaling responses in epithelial cells and may have broader implications in EBOV pathogenesis.

Antibody quality and protection from lethal Ebola virus challenge in nonhuman primates immunized with rabies virus based bivalent vaccine.

We have previously described the generation of a novel Ebola virus (EBOV) vaccine platform based on (a) replication-competent rabies virus (RABV), (b) replication-deficient RABV, or (c) chemically inactivated RABV expressing EBOV glycoprotein (GP). Mouse studies demonstrated safety, immunogenicity, and protective efficacy of these live or inactivated RABV/EBOV vaccines. Here, we evaluated these vaccines in nonhuman primates. Our results indicate that all three vaccines do induce potent immune responses against both RABV and EBOV, while the protection of immunized animals against EBOV was largely dependent on the quality of humoral immune response against EBOV GP. We also determined if the induced antibodies against EBOV GP differ in their target, affinity, or the isotype. Our results show that IgG1-biased humoral responses as well as high levels of GP-specific antibodies were beneficial for the control of EBOV infection after immunization. These results further support the concept that a successful EBOV vaccine needs to induce strong antibodies against EBOV. We also showed that a dual vaccine against RABV and filoviruses is achievable; therefore addressing concerns for the marketability of this urgently needed vaccine.

Systems kinomics demonstrates Congo Basin monkeypox virus infection selectively modulates host cell signaling responses as compared to West African monkeypox virus.

Monkeypox virus (MPXV) is comprised of two clades: Congo Basin MPXV, with an associated case fatality rate of 10%, and Western African MPXV, which is associated with less severe infection and minimal lethality. We thus postulated that Congo Basin and West African MPXV would differentially modulate host cell responses and, as many host responses are regulated through phosphorylation independent of transcription or translation, we employed systems kinomics with peptide arrays to investigate these functional host responses. Using this approach we have demonstrated that Congo Basin MPXV infection selectively down-regulates host responses as compared with West African MPXV, including growth factor- and apoptosis-related responses. These results were confirmed using fluorescence-activated cell sorting analysis demonstrating that West African MPXV infection resulted in a significant increase in apoptosis in human monocytes as compared with Congo Basin MPXV. Further, differentially phosphorylated kinases were identified through comparison of our MPXV data sets and validated as potential targets for pharmacological inhibition of Congo Basin MPXV infection, including increased Akt S473 phosphorylation and decreased p53 S15 phosphorylation. Inhibition of Akt S473 phosphorylation resulted in a significant decrease in Congo Basin MPXV virus yield (261-fold) but did not affect West African MPXV. In addition, treatment with staurosporine, an apoptosis activator resulted in a 49-fold greater decrease in Congo Basin MPXV yields as compared with West African MPXV. Thus, using a systems kinomics approach, our investigation demonstrates that West African and Congo Basin MPXV differentially modulate host cell responses and has identified potential host targets of therapeutic interest.

Intrabronchial inoculation of cynomolgus macaques with cowpox virus.

The public health threat of orthopoxviruses from bioterrorist attacks has prompted researchers to develop suitable animal models for increasing our understanding of viral pathogenesis and evaluation of medical countermeasures (MCMs) in compliance with the FDA Animal Efficacy Rule. We present an accessible intrabronchial cowpox virus (CPXV) model that can be evaluated under biosafety level-2 laboratory conditions. In this dose-ranging study, utilizing cynomolgus macaques, signs of typical orthopoxvirus disease were observed with the lymphoid organs, liver, skin (generally mild) and respiratory tract as target tissues. Clinical and histopathological evaluation suggests that intrabronchial CPXV recapitulated many of the features of monkeypox and variola virus, the causative agent of smallpox, infections in cynomolgus macaque models. These similarities suggest that CPXV infection in non-human primates should be pursued further as an alternative model of smallpox. Further development of the CPXV primate model, unimpeded by select agent and biocontainment restrictions, should facilitate the development of MCMs for smallpox.

Inactivated or live-attenuated bivalent vaccines that confer protection against rabies and Ebola viruses.

The search for a safe and efficacious vaccine for Ebola virus continues, as no current vaccine candidate is nearing licensure. We have developed (i) replication-competent, (ii) replication-deficient, and (iii) chemically inactivated rabies virus (RABV) vaccines expressing Zaire Ebola virus (ZEBOV) glycoprotein (GP) by a reverse genetics system based on the SAD B19 RABV wildlife vaccine. ZEBOV GP is efficiently expressed by these vaccine candidates and is incorporated into virions. The vaccine candidates were avirulent after inoculation of adult mice, and viruses with a deletion in the RABV glycoprotein had greatly reduced neurovirulence after intracerebral inoculation in suckling mice. Immunization with live or inactivated RABV vaccines expressing ZEBOV GP induced humoral immunity against each virus and conferred protection from both lethal RABV and EBOV challenge in mice. The bivalent RABV/ZEBOV vaccines described here have several distinct advantages that may speed the development of inactivated vaccines for use in humans and potentially live or inactivated vaccines for use in nonhuman primates at risk of EBOV infection in endemic areas.

Comparative analysis of monkeypox virus infection of cynomolgus macaques by the intravenous or intrabronchial inoculation route.

Monkeypox virus (MPXV) infection has recently expanded in geographic distribution and can be fatal in up to 10% of cases. The intravenous (i.v.) inoculation of nonhuman primates (NHPs) results in an accelerated fulminant disease course compared to that of naturally occurring MPXV infection in humans. Alternative routes of inoculation are being investigated to define an NHP model of infection that more closely resembles natural disease progression. Our goal was to determine if the intrabronchial (i.b.) exposure of NHPs to MPXV results in a systemic disease that better resembles the progression of human MPXV infection. Here, we compared the disease course following an i.v. or i.b. inoculation of NHPs with 10-fold serial doses of MPXV Zaire. Classical pox-like disease was observed in NHPs administered a high virus dose by either route. Several key events were delayed in the highest doses tested of the i.b. model compared to the timing of the i.v. model, including the onset of fever, lesion appearance, peak viremia, viral shedding in nasal and oral swabs, peak cytokine levels, and time to reach endpoint criteria. Virus distribution across 19 tissues was largely unaffected by the inoculation route at the highest doses tested. The NHPs inoculated by the i.b. route developed a viral pneumonia that likely exacerbated disease progression. Based on the observations of the delayed onset of clinical and virological parameters and endpoint criteria that may more closely resemble those of human MPXV infection, the i.b. MPXV model should be considered for the further investigation of viral pathogenesis and countermeasures.

Antiviral activity of the EB peptide against zoonotic poxviruses.

BACKGROUND: The EB peptide is a 20-mer that was previously shown to have broad spectrum in vitro activity against several unrelated viruses, including highly pathogenic avian influenza, herpes simplex virus type I, and vaccinia, the prototypic orthopoxvirus. To expand on this work, we evaluated EB for in vitro activity against the zoonotic orthopoxviruses cowpox and monkeypox and for in vivo activity in mice against vaccinia and cowpox. FINDINGS: In yield reduction assays, EB had an EC50 of 26.7 µM against cowpox and 4.4 µM against monkeypox. The EC50 for plaque reduction was 26.3 µM against cowpox and 48.6 µM against monkeypox. A scrambled peptide had no inhibitory activity against either virus. EB inhibited cowpox in vitro by disrupting virus entry, as evidenced by a reduction of the release of virus cores into the cytoplasm. Monkeypox was also inhibited in vitro by EB, but at the attachment stage of infection. EB showed protective activity in mice infected intranasally with vaccinia when co-administered with the virus, but had no effect when administered prophylactically one day prior to infection or therapeutically one day post-infection. EB had no in vivo activity against cowpox in mice. CONCLUSIONS: While EB did demonstrate some in vivo efficacy against vaccinia in mice, the limited conditions under which it was effective against vaccinia and lack of activity against cowpox suggest EB may be more useful for studying orthopoxvirus entry and attachment in vitro than as a therapeutic against orthopoxviruses in vivo.

Evaluation of monkeypox disease progression by molecular imaging.

Infection of nonhuman primates (NHPs) with monkeypox virus (MPXV) is currently being developed as an animal model of variola infection in humans. We used positron emission tomography and computed tomography (PET/CT) to identify inflammatory patterns as predictors for the outcome of MPXV disease in NHPs. Two NHPs were sublethally inoculated by the intravenous (IV) or intrabronchial (IB) routes and imaged sequentially using fluorine-18 fluorodeoxyglucose ((18)FDG) uptake as a nonspecific marker of inflammation/immune activation. Inflammation was observed in the lungs of IB-infected NHPs, and bilobular involvement was associated with morbidity. Lymphadenopathy and immune activation in the axillary lymph nodes were evident in IV- and IB-infected NHPs. Interestingly, the surviving NHPs had significant (18)FDG uptake in the axillary lymph nodes at the time of MPXV challenge with no clinical signs of illness, suggesting an association between preexisting immune activation and survival. Molecular imaging identified patterns of inflammation/immune activation that may allow risk assessment of monkeypox disease.

Targeted mutagenesis as a rational approach to dengue virus vaccine development.

The recombinant dengue virus type 4 (rDEN4) vaccine candidate, rDEN4Delta30, was found to be highly infectious, immunogenic and safe in human volunteers. At the highest dose (10(5) PFU) evaluated in volunteers, 25% of the vaccinees had mild elevations in liver enzymes that were rarely seen at lower doses. Here, we describe the generation and selection of additional mutations that were introduced into rDEN4Delta30 to further attenuate the virus in animal models and ultimately human vaccinees. Based on the elevated liver enzymes associated with the 10(5) PFU dose of rDEN4Delta30 and the known involvement of liver infection in dengue virus pathogenesis, a large panel of mutant viruses was screened for level of replication in the HuH-7 human hepatoma cell line, a surrogate for human liver cells and selected viruses were further analyzed for level of viremia in SCID-HuH-7 mice. It was hypothesized that rDEN4Delta30 derivatives with restricted replication in vitro and in vivo in HuH-7 human liver cells would be restricted in replication in the liver of vaccinees. Two mutations identified by this screen, NS3 4995 and NS5 200,201, were separately introduced into rDEN4Delta30 and found to further attenuate the vaccine candidate for SCID-HuH-7 mice and rhesus monkeys while retaining sufficient immunogenicity in rhesus monkeys to confer protection. In humans, the rDEN4Delta30-200,201 vaccine candidate administered at 10(5) PFU exhibited greatly reduced viremia, high infectivity and lacked liver toxicity while inducing serum neutralizing antibody at a level comparable to that observed in volunteers immunized with rDEN4Delta30. Clinical studies of rDEN4Delta30-4995 are ongoing.

Vaccine candidates for dengue virus type 1 (DEN1) generated by replacement of the structural genes of rDEN4 and rDEN4Delta30 with those of DEN1.

BACKGROUND: Antigenic chimeric viruses have previously been generated in which the structural genes of recombinant dengue virus type 4 (rDEN4) have been replaced with those derived from DEN2 or DEN3. Two vaccine candidates were identified, rDEN2/4Delta30(ME) and rDEN3/4Delta30(ME), which contain the membrane (M) precursor and envelope (E) genes of DEN2 and DEN3, respectively, and a 30 nucleotide deletion (Delta30) in the 3' untranslated region of the DEN4 backbone. Based on the promising preclinical phenotypes of these viruses and the safety and immunogenicity of rDEN2/4Delta30(ME) in humans, we now describe the generation of a panel of four antigenic chimeric DEN4 viruses using either the capsid (C), M, and E (CME) or ME structural genes of DEN1 Puerto Rico/94 strain. RESULTS: Four antigenic chimeric viruses were generated and found to replicate efficiently in Vero cells: rDEN1/4(CME), rDEN1/4Delta30(CME), rDEN1/4(ME), and rDEN1/4Delta30(ME). With the exception of rDEN1/4(ME), each chimeric virus was significantly attenuated in a SCID-HuH-7 mouse xenograft model with a 25-fold or greater reduction in replication compared to wild type DEN1. In rhesus monkeys, only chimeric viruses with the Delta30 mutation appeared to be attenuated as measured by duration and magnitude of viremia. rDEN1/4Delta30(CME) appeared over-attenuated since it failed to induce detectable neutralizing antibody and did not confer protection from wild type DEN1 challenge. In contrast, rDEN1/4Delta30(ME) induced 66% seroconversion and protection from DEN1 challenge. Presence of the Delta30 mutation conferred a significant restriction in mosquito infectivity upon rDEN1/4Delta30(ME) which was shown to be non-infectious for Aedes aegypti fed an infectious bloodmeal. CONCLUSION: The attenuation phenotype in SCID-HuH-7 mice, rhesus monkeys, and mosquitoes and the protective immunity observed in rhesus monkeys suggest that rDEN1/4Delta30(ME) should be considered for evaluation in a clinical trial.

Development of a live attenuated dengue virus vaccine using reverse genetics.

There are four serotypes of dengue (DEN1-DEN4) virus that are endemic in most areas of Southeast Asia, Central and South America, and other subtropical regions. The number of cases of severe disease associated with DEN virus infection is growing because of the continued spread of the mosquito vector, Aedes aegypti, which transmits the virus to humans. Infection with DEN virus can result in an asymptomatic infection, a febrile illness called dengue fever (DF), and the very severe disease called dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Currently, a licensed vaccine is not available. However, a tetravalent vaccine is urgently needed to prevent DF and DHF/DSS, the latter of which occurs predominantly in partially immune individuals. A live attenuated, tetravalent DEN virus vaccine candidate has been generated using reverse genetics that is able to provide immunity to each of the four serotypes of DEN. Attenuation has been achieved by generating recombinant DEN (rDEN) viruses which are modified by deletion or, alternatively, by antigenic chimerization between two related DEN viruses using the following two strategies: 1) introduction of an attenuating 30 nucleotide deletion (Delta30) mutation into the 3' untranslated region of DEN1 and DEN4; and 2) replacement of structural proteins of the attenuated rDEN4Delta30 vaccine candidate with those from DEN2 or DEN3. Attenuation of the four monovalent vaccine candidates has been achieved for rhesus monkeys or humans and an immunogenic tetravalent vaccine candidate has been formulated. The level of attenuation of each dengue vaccine component can be increased, if needed, by introduction of additional attenuating mutations that have been well characterized.

Controlled viral glycoprotein expression as a safety feature in a bivalent rabies-ebola vaccine.

Using a recombinant rabies (RABV) vaccine platform, we have developed several safe and effective vaccines. Most recently, we have developed a RABV-based ebolavirus (EBOV) vaccine that is efficacious in nonhuman primates. One safety feature of this vaccine is the utilization of a live but replication-deficient RABV construct. In this construct, the RABV glycoprotein (G) has been deleted from the genome, requiring G trans complementation in order for new infectious viruses to be released from the initial infected cell. Here we analyze this safety feature of the bivalent RABV-based EBOV vaccine comprised of the G-deleted RABV backbone expressing EBOV glycoprotein (GP). We found that, while the level of RABV genome in infected cells is equivalent regardless of G supplementation, the production of infectious virus is indeed restricted by the lack of G, and most importantly, that the presence of EBOV GP does not substitute for G. These findings further support the safety profile of this replication-deficient RABV-EBOV bivalent vaccine.

Small particle aerosol inoculation of cowpox Brighton Red in rhesus monkeys results in a severe respiratory disease.

Cowpox virus (CPXV) inoculation of nonhuman primates (NHPs) has been suggested as an alternate model for smallpox (Kramski et al., 2010, PLoS One, 5, e10412). Previously, we have demonstrated that intrabronchial inoculation of CPXV-Brighton Red (CPXV-BR) into cynomolgus monkeys resulted in a disease that shared many similarities to smallpox; however, severe respiratory tract disease was observed (Smith et al., 2011, J. Gen. Virol.). Here we describe the course of disease after small particle aerosol exposure of rhesus monkeys using computed tomography (CT) to monitor respiratory disease progression. Subjects developed a severe respiratory disease that was uniformly lethal at 5.7 log10 PFU of CPXV-BR. CT indicated changes in lung architecture that correlated with changes in peripheral blood monocytes and peripheral oxygen saturation. While the small particle aerosol inoculation route does not accurately mimic human smallpox, the data suggest that CT can be used as a tool to monitor real-time disease progression for evaluation of animal models for human diseases.

Genetically modified, live attenuated dengue virus type 3 vaccine candidates.

Three novel recombinant dengue type 3 (DEN3) virus vaccine candidates have been generated from a DEN3 virus isolated from a mild outbreak of dengue fever in the Sleman area of central Java in Indonesia in 1978. Antigenic chimeric viruses were prepared by replacing the membrane precursor and envelope (ME) proteins of recombinant DEN4 (rDEN4) virus with those from DEN3 Sleman/78 in the presence (rDEN3/4Delta30(ME)) and the absence (rDEN3/4(ME)) of the Delta30 mutation, a previously described 30-nucleotide deletion in the 3' untranslated region. In addition, a full-length infectious cDNA clone was generated from the DEN3 isolate and used to produce rDEN3 virus and the vaccine candidate rDEN3Delta30. The chimeric viruses rDEN3/4(ME) and rDEN3/4Delta30(ME) appear to be acceptable vaccine candidates since they were restricted in replication in severe combined immune deficiency mice transplanted with human hepatoma cells, in rhesus monkeys, and in Aedes and Toxorynchites mosquitoes, and each was protective in rhesus monkeys against DEN3 virus challenge. The rDEN3/4(ME) and rDEN3/4Delta30(ME) viruses were comparable in all parameters evaluated, indicating that antigenic chimerization resulted in the observed high level of attenuation. Surprisingly, rDEN3Delta30 was not attenuated in any model tested when compared with wild-type rDEN3 and therefore, is not a vaccine candidate at present. Thus, the rDEN3/4(ME) and rDEN3/4Delta30(ME) antigenic chimeric viruses can be considered for evaluation in humans and for inclusion in a tetravalent dengue vaccine.

Vaccine candidates derived from a novel infectious cDNA clone of an American genotype dengue virus type 2.

BACKGROUND: A dengue virus type 2 (DEN-2 Tonga/74) isolated from a 1974 epidemic was characterized by mild illness and belongs to the American genotype of DEN-2 viruses. To prepare a vaccine candidate, a previously described 30 nucleotide deletion (Delta30) in the 3' untranslated region of DEN-4 has been engineered into the DEN-2 isolate. METHODS: A full-length cDNA clone was generated from the DEN-2 virus and used to produce recombinant DEN-2 (rDEN-2) and rDEN2Delta30. Viruses were evaluated for replication in SCID mice transplanted with human hepatoma cells (SCID-HuH-7 mice), in mosquitoes, and in rhesus monkeys. Neutralizing antibody induction and protective efficacy were also assessed in rhesus monkeys. RESULTS: The rDEN2Delta30 virus was ten-fold reduced in replication in SCID-HuH-7 mice when compared to the parent virus. The rDEN-2 viruses were not infectious for Aedes mosquitoes, but both readily infected Toxorynchites mosquitoes. In rhesus monkeys, rDEN2Delta30 appeared to be slightly attenuated when compared to the parent virus as measured by duration and peak of viremia and neutralizing antibody induction. A derivative of rDEN2Delta30, designated rDEN2Delta30-4995, was generated by incorporation of a point mutation previously identified in the NS3 gene of DEN-4 and was found to be more attenuated than rDEN2Delta30 in SCID-HuH-7 mice. CONCLUSIONS: The rDEN2Delta30 and rDEN2Delta30-4995 viruses can be considered for evaluation in humans and for inclusion in a tetravalent dengue vaccine.

Matched longitudinal analysis of biomarkers associated with survival.

The identification of host or pathogen factors linked to clinical outcome is a common goal in many animal studies of infectious diseases. When the disease is fatal, statistical analysis of such factors may be biased from missing observations due to deaths. For example, when observations of a subject are censored before completing the intended study period, the complete trajectory will not be observed. Even if the factor is not associated with outcome, comparisons of data from survivors with those from nonsurvivors may lead to the wrong conclusions regarding associations with survival. Comparisons between subjects must account for differing observation lengths for those who survive relative to those who do not. Analyzing data over an interval common to all subjects provides one solution but requires eliminating data, some of which may be informative about the differences between groups. Here, we present a novel approach, matched longitudinal analysis (MLA), for analyzing such data based on matching biomarker intervals for survivors and nonsurvivors. We describe the results from simulation studies and from a study of monkeypox virus infection in nonhuman primates. In our application, MLA identified low monocyte chemoattractant protein-1 (MCP-1) levels as having a statistically significant association with survival, whereas the alternative methods did not identify an association. The method has general application to longitudinal studies that seek to find associations of biomarker changes with survival.

The effect of volume of interest definition on quantification of lymph node immune response to a monkeypox virus infection assessed by (18)F-FDG-PET.

BACKGROUND: 2-deoxy-2-[(18)F]fluoro-D-glucose-positron emission tomography ((18)F-FDG-PET) is applied in the clinic for infection assessment and is under consideration for investigating the inflammatory/immune response in lymphoid tissue in animal models of viral infection. Assessing changes in (18)F-FDG uptake of lymph nodes (LNs), primary lymphoid tissues targeted during viral infection, requires suitable methods for image analysis. Similar to tumor evaluation, reliable quantitation of the LN function via multiple (18)F-FDG-PET sessions will depend how the volume of interest is defined. Volume of interest definition has a direct effect on statistical outcome. The current study objective is to compare for the first time agreement between conventional and modified VOI metrics to determine which method(s) provide(s) reproducible standardized uptake values (SUVs) for (18)F-FDG uptake in the LN of rhesus macaques. METHODS: Multiple (18)F-FDG-PET images of LNs in macaques were acquired prior to and after monkeypox virus intravenous inoculation. We compared five image analysis approaches, SUVmax, SUVmean, SUVthreshold, modified SUVthreshold, and SUVfixed volume, to investigate the impact of these approaches on quantification of the changes in LN metabolic activity denoting the immune response during viral infection progression. RESULTS: The lowest data repeatability was observed with SUVmax. The best correspondence was between SUVfixed volume and conventional and modified SUVthreshold. A statistically significant difference in the LN (18)F-FDG uptake between surviving and moribund animals was shown using modified SUVthreshold and SUVfixed volume (adjusted p = 0.0037 and p = 0.0001, respectively). CONCLUSIONS: Quantification of the LN (18)F-FDG uptake is highly sensitive to the method applied for PET image analysis. SUVfixed volume and modified SUVthreshold demonstrate better reproducibility for SUV estimates than SUVmax, SUVmean, and SUVthreshold. SUVfixed volume and modified SUVthreshold are capable of distinguishing between groups with different disease outcomes. Therefore, these methods are the preferred approaches for evaluating the LN function during viral infection by (18)F-FDG-PET. Validation of multiple approaches is necessary to choose a suitable method to monitor changes in LN metabolic activity during progression of viral infection.