Last data update: Apr 29, 2024. (Total: 46658 publications since 2009)
Records 1-24 (of 24 Records) |
Query Trace: Roehrig JT[original query] |
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Genetic Adaptation by Dengue Virus Serotype 2 to Enhance Infection of Aedes aegypti Mosquito Midguts.
Erb SM , Butrapet S , Roehrig JT , Huang CY , Blair CD . Viruses 2022 14 (7) Dengue viruses (DENVs), serotypes 1-4, are arthropod-borne viruses transmitted to humans by mosquitoes, primarily Aedes aegypti. The transmission cycle begins when Ae. aegypti ingest blood from a viremic human and the virus infects midgut epithelial cells. In studying viruses derived from the DENV2 infectious clone 30P-NBX, we found that when the virus was delivered to female Ae. aegypti in an infectious blood meal, the midgut infection rate (MIR) was very low. To determine if adaptive mutations in the DENV2 envelope (E) glycoprotein could be induced to increase the MIR, we serially passed 30P-NBX in Ae. aegypti midguts. After four passages, a single, non-conservative mutation in E protein domain II (DII) nucleotide position 1300 became dominant, resulting in replacement of positively-charged amino acid lysine (K) at position 122 with negatively-charged glutamic acid (E; K122E) and a significantly-enhanced MIR. Site directed mutagenesis experiments showed that reducing the positive charge of this surface-exposed region of the E protein DII correlated with improved Ae. aegypti midgut infection. |
Exposing cryptic epitopes on the Venezuelan equine encephalitis virus E1 glycoprotein prior to treatment with alphavirus cross-reactive monoclonal antibody allows blockage of replication early in infection
Calvert AE , Bennett SL , Hunt AR , Fong RH , Doranz BJ , Roehrig JT , Blair CD . Virology 2021 565 13-21 Eastern equine encephalitis virus (EEEV), western equine encephalitis virus (WEEV) and Venezuelan equine encephalitis virus (VEEV) can cause fatal encephalitis in humans and equids. Some MAbs to the E1 glycoprotein are known to be cross-reactive, weakly neutralizing in vitro but can protect from disease in animal models. We investigated the mechanism of neutralization of VEEV infection by the broadly cross-reactive E1-specific MAb 1A4B-6. 1A4B-6 protected 3-week-old Swiss Webster mice prophylactically from lethal VEEV challenge. Likewise, 1A4B-6 inhibited virus growth in vitro at a pre-attachment step after virions were incubated at 37 °C and inhibited virus-mediated cell fusion. Amino acid residue N100 in the fusion loop of E1 protein was identified as critical for binding. The potential to elicit broadly cross-reactive MAbs with limited virus neutralizing activity in vitro but that can inhibit virus entry and protect animals from infection merits further exploration for vaccine and therapeutic developmental research. |
A monoclonal antibody specific for japanese encephalitis virus with high neutralizing capability for inclusion as a positive control in diagnostic neutralization tests
Calvert AE , Bennett SL , Dixon KL , Blair CD , Roehrig JT . Am J Trop Med Hyg 2019 101 (1) 233-236 Japanese encephalitis virus (JEV) is the most common cause of viral encephalitis in Asia, and it is increasingly a global public health concern because of its recent geographic expansion. Although commercial vaccines are available and used in some endemic countries, JEV continues to cause illness, with more than 60,000 cases reported annually. To develop a reproducible positive control antibody useable in diagnosis of JEV infections, murine hybridomas were developed from mice inoculated with a combination of IXIARO JEV vaccine and JEV domain III of the envelope protein (E-DIII). Monoclonal antibodies (MAbs) were characterized for their ability to neutralize virus in vitro. Monoclonal antibody 17BD3-2 was found to be JEV specific and highly neutralizing, with a plaque reduction neutralization test (PRNT)90 endpoint titer of 1.25 mug/mL. The functional epitopes were mapped using virus neutralization escape variants to amino acid residues S309, K312, and G333 in E-DIII. This MAb may be substituted for human immune sera used as a positive control in PRNT for distribution to public health laboratories worldwide in potential future outbreaks of JEV. |
A humanized monoclonal antibody neutralizes yellow fever virus strain 17D-204 in vitro but does not protect a mouse model from disease
Calvert AE , Dixon KL , Piper J , Bennett SL , Thibodeaux BA , Barrett AD , Roehrig JT , Blair CD . Antiviral Res 2016 131 92-9 The yellow fever virus (YFV) vaccine 17D-204 is considered safe and effective, yet rare severe adverse events (SAEs), some resulting in death, have been documented following vaccination. Individuals exhibiting post-vaccinal SAEs are ideal candidates for antiviral monoclonal antibody (MAb) therapy; the time until appearance of clinical signs post-exposure is usually short and patients are quickly hospitalized. We previously developed a murine-human chimeric monoclonal antibody (cMAb), 2C9-cIgG, reactive with both virulent YFV and 17D-204, and demonstrated its ability to prevent and treat YF disease in both AG129 mouse and hamster models of infection. To counteract possible selection of 17D-204 variants that escape neutralization by treatment with a single MAb (2C9-cIgG), we developed a second cMAb, 864-cIgG, for use in combination with 2C9-cIgG in post-vaccinal therapy. MAb 864-cIgG recognizes/neutralizes only YFV 17D-204 vaccine substrain and binds to domain III (DIII) of the viral envelope protein, which is different from the YFV type-specific binding site of 2C9-cIgG in DII. Although it neutralized 17D-204 in vitro, administration of 864-cIgG had no protective capacity in the interferon receptor-deficient AG129 mouse model of 17D-204 infection. The data presented here show that although DIII-specific 864-cIgG neutralizes virus infectivity in vitro, it does not have the ability to abrogate disease in vivo. Therefore, combination of 864-cIgG with 2C9-cIgG for treatment of YF vaccination SAEs does not appear to provide an improvement on 2C9-cIgG therapy alone. |
Locking and blocking the viral landscape of an alphavirus with neutralizing antibodies
Porta J , Jose J , Roehrig JT , Blair CD , Kuhn RJ , Rossmann MG . J Virol 2014 88 (17) 9616-23 Alphaviruses can be serious, sometimes lethal human pathogens that belong to the family Togaviridae. Structures of human Venezuelan equine encephalitis virus (VEEV), an alphavirus, in complex with two strongly neutralizing antibody Fab fragments (F5 and 3B4C-4) have been determined using a combination of cryo-electron microscopy (cryo-EM) and homology modeling. Here we characterize these monoclonal antibody Fab fragments known to abrogate VEEV infectivity by binding to the E2 (envelope) surface glycoprotein. Both these antibody Fab fragments cross-link the surface E2 glycoproteins and, therefore, probably inhibit infectivity by blocking the conformational changes that are required for making the virus fusogenic. The F5 Fab fragment cross-links E2 proteins within one trimeric spike, whereas the 3B4C-4 Fab fragment cross-links E2 proteins from neighboring spikes. Furthermore, F5 probably blocks the receptor-binding site, whereas 3B4C-4 sterically hinders the exposure of the fusion loop at the end of the E2 B-domain. IMPORTANCE: Alphaviral infections are transmitted mainly by mosquitoes. Venezuelan equine encephalitis virus (VEEV) is an alphavirus with a wide distribution across the globe. No effective vaccines exist for alphaviral infections. Therefore, a better understanding of VEEV and its associated neutralizing antibodies will help with the development of effective drugs and vaccines. |
Molecular determinants of dengue virus 2 envelope protein important for virus entry in Fc?RIIA-mediated antibody-dependent enhancement of infection.
Chotiwan N , Roehrig JT , Schlesinger JJ , Blair CD , Huang CYH . Virology 2014 456-457 (1) 238-246 Antibody-dependent enhancement (ADE) of infection may cause severe illness in patients suffering a secondary infection by a heterologous dengue virus (DENV) serotype. During ADE of infection, cross-reactive non- or poorly-neutralizing antibodies form infectious virus-Ab complexes with the newly infecting serotype and enhance virus infection by binding to the Fc receptors (FcR) on FcR-bearing cells. In this study, we determined that molecular determinants of DENV2 envelope protein critical for virus entry during non-ADE infection are also required for ADE infection mediated by FcRIIA, and binding of virus-Ab complexes with FcRIIA alone is not sufficient for ADE of infection. The FcRIIA mainly plays an auxiliary role in concentrating the virus-Ab complex to the cell surface, and other primary cellular receptors are required for virus entry. Understanding the viral entry pathway in ADE of DENV infection will greatly facilitate rational designs of anti-viral therapeutics against severe dengue disease associated with ADE. |
Humanized monoclonal antibody 2C9-cIgG has enhanced efficacy for yellow fever prophylaxis and therapy in an immunocompetent animal model
Julander JG , Thibodeaux BA , Morrey JD , Roehrig JT , Blair CD . Antiviral Res 2014 103c 32-38 Yellow fever virus (YFV) causes significant human disease and mortality in tropical regions of South and Central America and Africa, despite the availability of an effective vaccine. No specific therapy for YF is available. We previously showed that the humanized monoclonal antibody (MAb) 2C9-cIgG provided prophylactic and therapeutic protection from mortality in interferon receptor-deficient strain AG129 mice challenged with YF 17D-204 vaccine. In this study we tested the prophylactic and therapeutic efficacy of this MAb against virulent YFV infection in an immunocompetent hamster model. Intraperitoneal (ip) administration of a single dose of MAb 2C9-cIgG 24h prior to YFV challenge resulted in significantly improved survival rates in animals treated with 380 or 38mug of MAb compared to untreated animals. Treatment with the higher dose also resulted in significantly improved weight gain and reductions in serum alanine aminotransferase (ALT) and virus titers in serum and liver. Prophylactic treatment with 2C9-cIgG 24h prior to virus challenge prevented the development of a virus-neutralizing antibody (vnAb) response in hamsters. Administration of a single ip dose of 380mug of 2C9-cIgG as late as 72h post-YFV challenge also resulted in significant improvement in survival rates. Hamsters treated at 4-72h post-virus challenge developed a robust vnAb response. Enhanced survival and improvement of various disease parameters in the hamster model when MAb 2C9-cIgG is administered up to 3days after virus challenge demonstrate the clinical potential of specific antibody therapy for YF. |
West Nile virus in the United States - a historical perspective
Roehrig JT . Viruses 2013 5 (12) 3088-108 Prior to 1999, West Nile virus (WNV) was a bit player in the screenplay of global vector-borne viral diseases. First discovered in the West Nile District of Uganda in 1937, this Culex sp.-transmitted virus was known for causing small human febrile outbreaks in Africa and the Middle East. Prior to 1995, the last major human WNV outbreak was in the 1950s in Israel. The epidemiology and ecology of WNV began to change in the mid-1990s when an epidemic of human encephalitis occurred in Romania. The introduction of WNV into Eastern Europe was readily explained by bird migration between Africa and Europe. The movement of WNV from Africa to Europe could not, however, predict its surprising jump across the Atlantic Ocean to New York City and the surrounding areas of the United States (U.S.). This movement of WNV from the Eastern to Western Hemisphere in 1999, and its subsequent dissemination throughout two continents in less than ten years is widely recognized as one of the most significant events in arbovirology during the last two centuries. This paper documents the early events of the introduction into and the spread of WNV in the Western Hemisphere. |
Development of a small animal peripheral challenge model of Japanese encephalitis virus using interferon deficient AG129 mice and the SA14-14-2 vaccine virus strain
Calvert AE , Dixon KL , Delorey MJ , Blair CD , Roehrig JT . Vaccine 2013 32 (2) 258-64 Japanese encephalitis virus (JEV) is the most common cause of viral encephalitis in Asia, and it is increasingly a global public health concern due to its recent geographic expansion. While commercial vaccines are available and used in some endemic countries, JEV continues to be a public health problem, with 50,000 cases reported annually. Research with virulent JEV in mouse models to develop new methods of prevention and treatment is restricted to BSL-3 containment facilities, confining these studies to investigators with access to these facilities. We have developed an adult small animal peripheral challenge model using interferon-deficient AG129 mice and the JEV live-attenuated vaccine SA14-14-2, thus requiring only BSL-2 containment. A low dose of virus (10PFU/0.1ml) induced 100% morbidity in infected mice. Increased body temperatures measured by implantable temperature transponders correlated with an increase in infectious virus and viral RNA in serum, spleen and brain as well as an increase in pro-inflammatory markers measured by a 58-biomarker multi-analyte profile (MAP) constructed during the course of infection. In the future, the MAP measurements can be used as a baseline for comparison in order to better assess the inhibition of disease progression by other prophylactic and therapeutic agents. The use of the AG129/JEV SA14-14-2 animal model makes vaccine and therapeutic studies feasible for laboratories with limited biocontainment facilities. |
Long-term safety assessment of live attenuated tetravalent dengue vaccines: deliberations from a WHO technical consultation
Bentsi-Enchill AD , Schmitz J , Edelman R , Durbin A , Roehrig JT , Smith PG , Hombach J , Farrar J . Vaccine 2013 31 (23) 2603-9 Dengue is a rapidly growing public health threat with approximately 2.5 billion people estimated to be at risk. Several vaccine candidates are at various stages of pre-clinical and clinical development. Thus far, live dengue vaccine candidates have been administered to several thousands of volunteers and were well-tolerated, with minimal short-term safety effects reported in Phase I and Phase II clinical trials. Based on the natural history of dengue, a theoretical possibility of an increased risk of severe dengue as a consequence of vaccination has been hypothesized but not yet observed. In October 2011, the World Health Organization (WHO) convened a consultation of experts in dengue, vaccine regulation and vaccine safety to review the current scientific evidence regarding safety concerns associated with live attenuated dengue vaccines and, in particular, to consider methodological approaches for their long-term evaluation. In this paper we summarize the scientific background and methodological considerations relevant to the safety assessment of these vaccines. Careful planning and a coordinated approach to safety assessment are recommended to ensure adequate long-term evaluation of dengue vaccines that will support their introduction and continued use. |
Mutation of the dengue virus type 2 envelope protein heparan sulfate binding sites or the domain III lateral ridge blocks replication in Vero cells prior to membrane fusion.
Roehrig JT , Butrapet S , Liss NM , Bennett SL , Luy BE , Childers T , Boroughs KL , Stovall JL , Calvert AE , Blair CD , Huang CY . Virology 2013 441 (2) 114-25 Using an infectious cDNA clone we engineered seven mutations in the putative heparan sulfate- and receptor-binding motifs of the envelope protein of dengue virus serotype 2, strain 16681. Four mutant viruses, KK122/123EE, E202K, G304K, and KKK305/307/310EEE, were recovered following transfection of C6/36 cells. A fifth mutant, KK291/295EE, was recovered from C6/36 cells with a compensatory E295V mutation. All mutants grew in and mediated fusion of virus-infected C6/36 cells, but three of the mutants, KK122/123EE, E202K, G304K, did not grow in Vero cells without further modification. Two Vero cell lethal mutants, KK291/295EV and KKK307/307/310EEE, failed to replicate in DC-SIGN-transformed Raji cells and did not react with monoclonal antibodies known to block DENV attachment to Vero cells. Additionally, both mutants were unable to initiate negative-strand vRNA synthesis in Vero cells by 72h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion. |
Mutations in the West Nile prM protein affect VLP and virion secretion in vitro.
Calvert AE , Huang CY , Blair CD , Roehrig JT . Virology 2012 433 (1) 35-44 Mutation of the West Nile virus-like particle (WN VLP) prM protein (T20D, K31A, K31V, or K31T) results in undetectable VLP secretion from transformed COS-1 cells. K31 mutants formed intracellular prM-E heterodimers; however these proteins remained in the ER and ER-Golgi intermediary compartments of transfected cells. The T20D mutation affected glycosylation, heterodimer formation, and WN VLP secretion. When infectious viruses bearing the same mutations were used to infect COS-1 cells, K31 mutant viruses exhibited delayed growth and reduced infectivity compared to WT virus. Epitope maps of WN VLP and WNV prM were also different. These results suggest that while mutations in the prM protein can reduce or eliminate secretion of WN VLPs, they have less effect on virus. This difference may be due to the quantity of prM in WN VLPs compared to WNV or to differences in maturation, structure, and symmetry of these particles. |
A humanized IgG but not IgM antibody is effective in prophylaxis and therapy of yellow fever infection in an AG129/17D-204 peripheral challenge mouse model
Thibodeaux BA , Garbino NC , Liss NM , Piper J , Schlesinger JJ , Blair CD , Roehrig JT . Antiviral Res 2012 94 (1) 1-8 Yellow fever virus (YFV), a member of the genus Flavivirus, is a mosquito-borne virus found in tropical regions of Africa and South America that causes severe hepatic disease and death in humans. Despite the availability of effective vaccines, YFV is responsible for an estimated 200,000 cases and 30,000 deaths annually. There are currently no prophylactic or therapeutic strategies approved for use in human YFV infections. Furthermore, implementation of YFV 17D-204 vaccination campaigns has become problematic due to an increase in reported post-vaccinal adverse events. We have created human/murine chimeric MAbs of a YFV-reactive murine monoclonal antibody (mMAb), 2C9, that was previously shown to protect mice from lethal YFV infection and to have therapeutic activity. The new chimeric (cMAbs) were constructed by fusion of the m2C9 IgG gene variable regions with the constant regions of human IgG and IgM and expressed in Sp2 murine myelomas. The 2C9 cMAbs (2C9-cIgG and 2C9-cIgM) reacted with 17D-204 vaccine strain in an enzyme-linked immunosorbent assay and neutralized virus in vitro similarly to the parent m2C9. Both m2C9 and 2C9-cIgG when administered prophylactically 24h prior to infection protected AG129 mice from peripheral 17D-204 challenge at antibody concentrations 1.27mcg/mouse; however, the 2C9-cIgM did not protect even at a dose of 127mcg/mouse. The 17D-204 infection of AG129 mice is otherwise uniformly lethal. While the m2C9 was shown previously to be therapeutically effective in YFV-infected BALB/c mice at day 4 post-infection, the m2C9 and 2C9-cIgG demonstrated therapeutic activity only when administered 1 day post-infection in 17D-204-infected AG129 mice. |
A small animal peripheral challenge model of yellow fever using interferon-receptor deficient mice and the 17D-204 vaccine strain
Thibodeaux BA , Garbino NC , Liss NM , Piper J , Blair CD , Roehrig JT . Vaccine 2012 30 (21) 3180-7 Yellow fever virus (YFV), a member of the genus Flavivirus, is a mosquito-borne pathogen that requires wild-type (wt), virulent strains to be handled at biosafety level (BSL) 3, with HEPA-filtration of room air exhaust (BSL3+). YFV is found in tropical regions of Africa and South America and causes severe hepatic disease and death in humans. Despite the availability of effective vaccines (17D-204 or 17DD), YFV is still responsible for an estimated 200,000 cases of illness and 30,000 deaths annually. Besides vaccination, there are no other prophylactic or therapeutic strategies approved for use in human YF. Current small animal models of YF require either intra-cranial inoculation of YF vaccine to establish infection, or use of wt strains (e.g., Asibi) in order to achieve pathology. We have developed and characterized a BSL2, adult mouse peripheral challenge model for YFV infection in mice lacking receptors for interferons alpha, beta, and gamma (strain AG129). Intraperitoneal challenge of AG129 mice with 17D-204 is a uniformly lethal in a dose-dependent manner, and 17D-204-infected AG129 mice exhibit high viral titers in both brain and liver suggesting this infection is both neurotropic and viscerotropic. Furthermore the use of a mouse model permitted the construction of a 59-biomarker multi-analyte profile (MAP) using samples of brain, liver, and serum taken at multiple time points over the course of infection. This MAP serves as a baseline for evaluating novel therapeutics and their effect on disease progression. Changes (4-fold or greater) in serum and tissue levels of pro- and anti-inflammatory mediators as well as other factors associated with tissue damage were noted in AG129 mice infected with 17D-204 as compared to mock-infected control animals. |
Recommendations for publication of viral genetic data and sample access for novel viruses and strains.
Arrigo NC , Briese T , Calisher CH , Drebot MA , Hjelle B , Leduc JW , Powers AM , Repik PM , Roehrig JT , Schmaljohn CS , Tesh RB , Weaver SC . Am J Trop Med Hyg 2012 86 (2) 189-191 Reference collections of viruses and virus strains representing their temporal, geographic, and phenotypic ranges are critical to basic research and public health. Such collections have proved essential for helping to determine the sources of new outbreaks as well as studying viral pathogenesis, taxonomy, emergence, and evolution. The development and validation of new diagnostics, therapeutics, and vaccines with appropriate breadth of coverage also rely on comprehensive collections of virus strains for validation studies. Despite their critical importance, many reference collections now struggle to maintain contemporary virus isolates from across geographic and host ranges. As stated by Robert Shope, who for many years, maintained the World Reference Center on Arboviruses at Yale University and later at the University of Texas Medical Branch, “Virus collection has virtually ceased. We need to find a politically acceptable way to return to the collecting business, perhaps in the name of basic science or preservation of biological diversity.”1 This trend is the result of reduced virus isolation efforts and the regulatory burdens that many countries now require to share or transfer certain viruses to appropriate repositories. For example, in the United States, the U.S. Department of Agriculture (USDA), Centers for Disease Control and Prevention (CDC), and sometimes Convention on International Trade in Endangered Species (CITES) of Wild Fauna and Flora permits are required for the importation and/or transfer of many viruses or even host and vector samples; in addition, Commerce Department permits are required to export many viruses, sometimes even to endemic countries of their origin. For regulated select agents, an extra layer of permitting and security is involved. Delays caused by these permitting and compliance processes can have devastating consequences if they impact the exchange of materials needed to aid in the research and public health responses to disease outbreaks. |
Development of a human-murine chimeric immunoglobulin M for use in the serological detection of human alphavirus antibodies
Thibodeaux BA , Liss NM , Panella AN , Roehrig JT . Clin Vaccine Immunol 2011 18 (12) 2181-2 Diagnosis of human alphaviral infections relies on serological techniques such as the immunoglobulin M antibody capture enzyme-linked immunosorbent assay (MAC-ELISA). We have humanized the broadly alphavirus cross-reactive murine monoclonal antibody 1A4B-6 to create a reagent capable of replacing human positive sera in the MAC-ELISA for diagnosis of human alphaviral infections. |
Treatment of mice with human monoclonal antibody 24h after lethal aerosol challenge with virulent Venezuelan equine encephalitis virus prevents disease but not infection
Hunt AR , Bowen RA , Frederickson S , Maruyama T , Roehrig JT , Blair CD . Virology 2011 414 (2) 146-52 We recently described a Venezuelan equine encephalitis virus (VEEV)-specific human monoclonal antibody (MAb), F5 nIgG, that recognizes a new neutralization epitope on the VEEV E2 envelope glycoprotein. In this study, we investigated the ability of F5 nIgG given prophylactically or therapeutically to protect mice from subcutaneous or aerosolized VEEV infection. F5 nIgG had potent ability to protect mice from infection by either route when administered 24h before exposure; however, mice treated 24h after aerosol exposure developed central nervous system infections but exhibited no clinical signs of disease. Infectious virus, viral antigen and RNA were detected in brains of both treated and untreated mice 2-6days after aerosol exposure but were cleared from the brains of treated animals by 14-28days after infection. This fully human MAb could be useful for prophylaxis or immediate therapy for individuals exposed to VEEV accidentally in the laboratory or during a deliberate release. |
Amino acid changes within the E protein hinge region that affect dengue virus type 2 infectivity and fusion
Butrapet S , Childers T , Moss KJ , Erb SM , Luy BE , Calvert AE , Blair CD , Roehrig JT , Huang CY . Virology 2011 413 (1) 118-27 Fifteen mutant dengue viruses were engineered and used to identify AAs in the molecular hinge of the envelope protein that are critical to viral infection. Substitutions at Q52, A54, or E133 reduced infectivity in mammalian cells and altered the pH threshold of fusion. Mutations at F193, G266, I270, or G281 affected viral replication in mammalian and mosquito cells, but only I270W had reduced fusion activity. T280Y affected the pH threshold for fusion and reduced replication in C6/36 cells. Three different mutations at L135 were lethal in mammalian cells. Among them, L135G abrogated fusion and reduced replication in C6/36 cells, but only slightly reduced the mosquito infection rate. Conversely, L135W replicated well in C6/36 cells, but had the lowest mosquito infection rate. Possible interactions between hinge residues 52 and 277, or among 53, 135, 170, 186, 265, and 276 required for hinge function were discovered by sequence analysis to identify compensatory mutations. |
Human monoclonal antibodies to West Nile virus identify epitopes on the prM protein.
Calvert AE , Kalantarov GF , Chang GJ , Trakht I , Blair CD , Roehrig JT . Virology 2011 410 (1) 30-7 Hybridoma cell lines (2E8, 8G8 and 5G12) producing fully human monoclonal antibodies (hMAbs) specific for the pre-membrane (prM) protein of West Nile virus (WNV) were prepared using a human fusion partner cell line, MFP-2, and human peripheral blood lymphocytes from a blood donor diagnosed with WNV fever in 2004. Using site-directed mutagenesis of a WNV-like particle (VLP) we identified 4 amino acid residues in the prM protein unique to WNV and important in the binding of these hMAbs to the VLP. Residues V19 and L33 are important epitopes for the binding of all three hMAbs. Mutations at residue, T20 and T24 affected the binding of hMAbs, 8G8 and 5G12 only. These hMAbs did not significantly protect AG129 interferon-deficient mice or Swiss Webster outbred mice from WNV infection. |
Alphaviruses
Powers AM , Roehrig JT . Methods Mol Biol 2011 665 17-38 Alphaviruses remain important emerging mosquito-borne, zoonotic pathogens that cause both localized human outbreaks and epizootics (e.g., Venezuelan equine encephalitis) and large human epidemics (e.g., Chikungunya). Alphaviruses are globally dispersed, and each continent has humans at risk from one or more of these arthropod-borne viruses (arboviruses). Symptoms of human alphaviral disease range from frank, severe encephalitis (e.g., eastern and western equine encephalitis) to polyarthritis (e.g., Ross River). Diagnostic techniques to identify human alphaviral infections have changed dramatically with the development and implementation of standardized nucleic acid amplification tests (NAAT). The NAAT is rapidly replacing virus isolation and typing using indirect fluorescent antibody (IFA) assay with monoclonal antibodies (MAbs) as the preferred method of virus identification. The older techniques still have value, however, since alphaviral growth in cell culture is rapid, and IFA with MAbs is inexpensive. This chapter provides detailed, standardized protocols for the identification of alphaviruses from clinical specimens and the serological characterization of human infection-immune sera. Both laboratory approaches are needed to identify and confirm human infections with these agents. |
Mutations of an antibody binding energy hot spot on domain III of the dengue 2 envelope glycoprotein exploited for neutralization escape
Gromowski GD , Roehrig JT , Diamond MS , Lee JC , Pitcher TJ , Barrett AD . Virology 2010 407 (2) 237-46 Previous crystallographic studies have identified a total of 11 DENV-2 envelope protein domain III (ED3) residues (K305, F306, K307, V308, V309, K310, I312, Q325, P364, K388, and N390) that interacted, through both side- and main-chain contacts, with the Fab of a dengue virus (DENV) subcomplex-specific neutralizing monoclonal antibody (MAb) 1A1D-2 (Lok et al., 2008). Here, we used DENV-2 recombinant ED3 mutants of the MAb 1A1D-2 structural epitope residues to determine the functional epitope of this MAb. The side-chains of residues K307, K310 and I312 were determined to be functionally critical for MAb binding, and thus constitute a hot spot of binding energy for MAb 1A1D-2 on the DENV-2 ED3. Overall, these findings demonstrate that only a subset of the amino acid residue side-chains within the structural epitope of MAb 1A1D-2 define a functional epitope on the DENV-2 ED3 that is essential for MAb binding and neutralization escape. |
Domain-III FG loop of the dengue virus type 2 envelope protein is important for infection of mammalian cells and Aedes aegypti mosquitoes
Erb SM , Butrapet S , Moss KJ , Luy BE , Childers T , Calvert AE , Silengo SJ , Roehrig JT , Huang CY , Blair CD . Virology 2010 406 (2) 328-35 The FG extended loop in domain III of the dengue virus type 2 (DENV2) envelope protein is postulated to be a molecular determinant for host cell infectivity. To determine the contribution of the FG loop to virus infectivity, an infectious cDNA clone of DENV2 was manipulated by deleting amino acids in the loop (VEPGDelta) to mimic tick-borne flaviviruses or by substituting these AAs with RGD or RGDK/S to mimic motifs present in other mosquito-borne flaviviruses. We found the FG loop to be dispensable for infection of C6/36 cells but critical for infection of Aedes aegypti mosquito midguts and mammalian cells. All the FG loop mutants were able to bind to and enter mammalian cells but replication of VEPGDelta in Vero cells at 37 degrees C was delayed until acquisition of secondary mutations. Reduced binding of DENV2 type-specific monoclonal antibody 3H5 to mutant viruses confirmed the FG loop motif as its target epitope. |
Development of human-murine chimeric immunoglobulin Gs for use in the serological detection of human flavivirus and alphavirus antibodies
Thibodeaux BA , Panella AN , Roehrig JT . Clin Vaccine Immunol 2010 17 (10) 1617-23 Diagnosis of human arboviral infections relies heavily on serological techniques such as the immunoglobulin (Ig) M antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) and the indirect IgG ELISA. Broad application of these assays is hindered by the lack of standardized positive human control sera that react with the wide variety of flaviviruses (e.g., dengue [DEN], West Nile [WN], yellow fever [YF], Japanese encephalitis [JE], Saint Louis encephalitis [SLE], and Powassan [POW] viruses), or alphaviruses (e.g., Eastern equine encephalitis [EEE], Western equine encephalitis [WEE], Venezuelan equine encephalitis [VEE], and chikungunya [CHIK] viruses) that can cause human disease. We have created human-murine chimeric monoclonal antibodies (cMAbs) by combining the variable regions of broadly flavivirus (6B6C-1) or alphavirus (1A4B-6) cross-reactive murine MAbs (mMAbs) with the constant region of human IgG1. These cMAbs may be used as standardized reagents capable of replacing human positive infection-immune control sera in indirect IgG ELISA for diagnosis of all human flavi- or alphaviral infections. The IgG cMAbs secreted from plasmid-transformed Sp2/0-Ag14 cells had serological activity identical to the parent mMAbs as measured by ELISA using multiple flaviviruses or alphaviruses. |
The dengue virus type 2 envelope protein fusion peptide is essential for membrane fusion
Huang CY , Butrapet S , Moss KJ , Childers T , Erb SM , Calvert AE , Silengo SJ , Kinney RM , Blair CD , Roehrig JT . Virology 2009 396 (2) 305-15 The flaviviral envelope (E) protein directs virus-mediated membrane fusion. To investigate membrane fusion as a requirement for virus growth, we introduced 27 unique mutations into the fusion peptide of an infectious cDNA clone of dengue 2 virus and recovered seven stable mutant viruses. The fusion efficiency of the mutants was impaired, demonstrating for the first time the requirement for specific FP AAs in optimal fusion. Mutant viruses exhibited different growth kinetics and/or genetic stabilities in different cell types and adult mosquitoes. Virus particles could be recovered following RNA transfection of cells with four lethal mutants; however, recovered viruses could not re-infect cells. These viruses could enter cells, but internalized virus appeared to be retained in endosomal compartments of infected cells, thus suggesting a fusion blockade. Mutations of the FP also resulted in reduced virus reactivity with flavivirus group-reactive antibodies, confirming earlier reports using virus-like particles. |
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