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Protective Cytotoxic T-Cell Responses Induced by Venezuelan Equine Encephalitis Virus Replicons Expressing Ebola Virus Proteins
Olinger, Gene G.
Bailey, Michael A.
Dye, John M.
Bakken, Russell
Kuehne, Ana
Kondig, John
Wilson, Julie
Hogan, Robert J.
Hart, Mary Kate
Journal of Virology
Infection with Ebola virus causes a severe disease accompanied by high mortality rates, and there are no licensed vaccines or therapies available for human use. Filovirus vaccine research efforts still need to determine the roles of humoral and cell-mediated immune responses in protection from Ebola virus infection. Previous studies indicated that exposure to Ebola virus proteins expressed from packaged Venezuelan equine encephalitis virus replicons elicited protective immunity in mice and that antibody-mediated protection could only be demonstrated after vaccination against the glycoprotein. In this study, the murine CD8+ T-cell responses to six Ebola virus proteins were examined. CD8+ T cells specific for Ebola virus glycoprotein, nucleoprotein, and viral proteins (VP24, VP30, VP35, and VP40) were identified by intracellular cytokine assays using splenocytes from vaccinated mice. The cells were expanded by restimulation with peptides and demonstrated cytolytic activity. Adoptive transfer of the CD8+ cytotoxic T cells protected filovirus naïve mice from challenge with Ebola virus. These data support a role for CD8+ cytotoxic T cells as part of a protective mechanism induced by vaccination against six Ebola virus proteins and provide additional evidence that cytotoxic T-cell responses can contribute to protection from filovirus infections.
Related Articles
Individual and Bivalent Vaccines Based on Alphavirus Replicons Protect Guinea Pigs against Infection with Lassa and Ebola Viruses
Pushko, Peter
Geisbert, Joan
Parker, Michael
Jahrling, Peter
Smith, Jonathan
Journal of Virology
Lassa and Ebola viruses cause acute, often fatal, hemorrhagic fever diseases, for which no effective vaccines are currently available. Although lethal human disease outbreaks have been confined so far to sub-Saharan Africa, they also pose significant epidemiological concern worldwide as demonstrated by several instances of accidental importation of the viruses into North America and Europe. In the present study, we developed experimental individual vaccines for Lassa virus and bivalent vaccines for Lassa and Ebola viruses that are based on an RNA replicon vector derived from an attenuated strain of Venezuelan equine encephalitis virus. The Lassa and Ebola virus genes were expressed from recombinant replicon RNAs that also encoded the replicase function and were capable of efficient intracellular self-amplification. For vaccinations, the recombinant replicons were incorporated into virus-like replicon particles. Guinea pigs vaccinated with particles expressing Lassa virus nucleoprotein or glycoprotein genes were protected from lethal challenge with Lassa virus. Vaccination with particles expressing Ebola virus glycoprotein gene also protected the animals from lethal challenge with Ebola virus. In order to evaluate a single vaccine protecting against both Lassa and Ebola viruses, we developed dual-expression particles that expressed glycoprotein genes of both Ebola and Lassa viruses. Vaccination of guinea pigs with either dual-expression particles or with a mixture of particles expressing Ebola and Lassa virus glycoprotein genes protected the animals against challenges with Ebola and Lassa viruses. The results showed that immune responses can be induced against multiple vaccine antigens coexpressed from an alphavirus replicon and suggested the possibility of engineering multivalent vaccines based upon alphavirus vectors for arenaviruses, filoviruses, and possibly other emerging pathogens.
Related Articles
Immune Protection of Nonhuman Primates against Ebola Virus with Single Low-Dose Adenovirus Vectors Encoding Modified GPs
Sullivan, Nancy J
Geisbert, Thomas W
Geisbert, Joan B
Shedlock, Devon J
Xu, Ling
Lamoreaux, Laurie
Custers, Jerome H. H. V
Popernack, Paul M
Yang, Zhi-Yong
Pau, Maria G
Roederer, Mario
Koup, Richard A
Goudsmit, Jaap
Jahrling, Peter B
Nabel, Gary J
PLoS Medicine
Ebola virus causes a hemorrhagic fever syndrome that is associated with high mortality in humans. In the absence of effective therapies for Ebola virus infection, the development of a vaccine becomes an important strategy to contain outbreaks. Immunization with DNA and/or replication-defective adenoviral vectors (rAd) encoding the Ebola glycoprotein (GP) and nucleoprotein (NP) has been previously shown to confer specific protective immunity in nonhuman primates. GP can exert cytopathic effects on transfected cells in vitro, and multiple GP forms have been identified in nature, raising the question of which would be optimal for a human vaccine.
Methods and Findings
To address this question, we have explored the efficacy of mutant GPs from multiple Ebola virus strains with reduced in vitro cytopathicity and analyzed their protective effects in the primate challenge model, with or without NP. Deletion of the GP transmembrane domain eliminated in vitro cytopathicity but reduced its protective efficacy by at least one order of magnitude. In contrast, a point mutation was identified that abolished this cytopathicity but retained immunogenicity and conferred immune protection in the absence of NP. The minimal effective rAd dose was established at 1010 particles, two logs lower than that used previously.
Expression of specific GPs alone vectored by rAd are sufficient to confer protection against lethal challenge in a relevant nonhuman primate model. Elimination of NP from the vaccine and dose reductions to 1010 rAd particles do not diminish protection and simplify the vaccine, providing the basis for selection of a human vaccine candidate.
A simplified Ebola vaccine that consists of a modified GP protein (which is well-tolerated by human cells even at high concentrations) in a replication-defective adenoviral vector protects macaques.
Editors' Summary
Humans who get infected with Ebola virus develop an illness called Ebola hemorrhagic fever (EHV), which is one of the most deadly viral diseases known; 50%–90% of all ill patients die, and there is no available treatment for EHV. Scientists think that the occasional outbreaks of the disease occur because the virus "jumps" from an infected animal to a person (a rare event) and then is transmitted between people by direct contact with infected blood or other body fluids or parts. Several strains or variants of the Ebola virus exist. Most outbreaks have been caused either by the Zaire strain or by the Sudan/Gulu strain (so-called because that is where the particular virus was first isolated). Scientists are working on a vaccine against Ebola that could be given to people before they get infected and then protect them when they come in contact with the virus. A number of candidate vaccines have been developed and tested in animals.
Why Was This Study Done?
The researchers who did this study are working on a vaccine that consists of two particular parts of the virus. One part is called GP (which stands for glycoprotein) and is from the outer coat of the virus; the other, NP (nucleoprotein), is from its inside. Without the rest of the virus, GP and NP cannot cause EBV. However, the hope is that giving these parts of the virus to an individual can educate their immune system to build a response against GP and NP, which would then recognize the virus should the vaccinated person become infected with the whole virus, and destroy it before it can cause disease. To get the GP and NP parts into the body so that they can cause a strong immune response (which is what effective vaccines do), the researchers used a manmade version of another, harmless virus called recombinant adenovirus 5 (or rAd5) to carry the NP and GP. The researchers have shown previously that this strategy for introducing a vaccine works in animals. The vaccine—i.e., the combination of the rAd5 virus and the two Ebola virus parts—can protect animals against subsequent infection with real Ebola virus that would otherwise kill them. However, during these earlier studies, the researchers had noticed that the GP part, when present at high levels, seemed to make human cells sick. They had not seen any similar problems in the experimental animals, but to be on the safe side they decided to see whether they could change the GP part so that it would still be effective as a vaccine but no longer make human cells sick.
What Did the Researchers Do and Find?
They changed the GP part of the vaccine in different ways so that it would no longer make human cells sick and then tested whether the resulting vaccines (combined with the original NP part and the Ad5 virus) could still protect monkeys from EHF after they were infected with Ebola virus. They found that some of the new GP versions made the vaccine less effective, but others did what they had hoped for; namely, they gave the same level of protection as when the original GP part was present. While doing these experiments, the researchers also found that the NP component seemed unnecessary and in some cases even weakened the vaccine's effect.
What Do These Findings Mean?
The researchers have now developed a simplified vaccine against Ebola virus that is effective in monkeys. This vaccine consists of only a modified GP component (which is well tolerated by human cells even at high concentrations) and the rAd5 component. This vaccine is not the only candidate currently being developed against Ebola, but it seems likely that it is one of a few that will be tested in human volunteers in the near future. The initial clinical trials will test whether the vaccine is safe in humans, and whether it can cause the immune system to produce an immune response that is specific for the Ebola virus. Assuming that the outcomes of these trials are positive, the next question is whether the vaccine can protect humans against Ebola disease. Because Ebola is so dangerous and outbreaks are relatively rare, the vaccine will likely be tested only during an actual outbreak. At that time, an experimental vaccine might be given to people at immediate risk of becoming infected, especially health-care workers who, because they take care of infected patients, are themselves at very high risk of becoming infected. In addition to trials in humans, the scientists will also explore whether this vaccine, which was developed based on the GP component of the Zaire strain, can protect monkeys against infections with other strains of the Ebola virus.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030177.
• World Health Organization
• MedlinePlus Medical Encyclopedia
• US Centers for Disease Control and Prevention
• Wikipedia (note: Wikipedia is a free Internet encyclopedia that anyone can edit)
Related Articles
Passive Transfer of Antibodies Protects Immunocompetent and Immunodeficient Mice against Lethal Ebola Virus Infection without Complete Inhibition of Viral Replication
Gupta, Manisha
Mahanty, Siddhartha
Bray, Mike
Ahmed, Rafi
Rollin, Pierre E.
Journal of Virology
Ebola hemorrhagic fever is a severe, usually fatal illness caused by Ebola virus, a member of the filovirus family. The use of nonhomologous immune serum in animal studies and blood from survivors in two anecdotal reports of Ebola hemorrhagic fever in humans has shown promise, but the efficacy of these treatments has not been demonstrated definitively. We have evaluated the protective efficacy of polyclonal immune serum in a mouse model of Ebola virus infection. Our results demonstrate that mice infected subcutaneously with live Ebola virus survive infection and generate high levels of anti-Ebola virus immunoglobulin G (IgG). Passive transfer of immune serum from these mice before challenge protected upto 100% of naive mice against lethal Ebola virus infection. Protection correlated with the level of anti-Ebola virus IgG titers, and passive treatment with high-titer antiserum was associated with a delay in the peak of viral replication. Transfer of immune serum to SCID mice resulted in 100% survival after lethal challenge with Ebola virus, indicating that antibodies alone can protect from lethal disease. Thus antibodies suppress or delay viral growth, provide protection against lethal Ebola virus infection, and may not require participation of other immune components for protection.
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Effective Post-Exposure Treatment of Ebola Infection
Feldmann, Heinz
Jones, Steven M
Daddario-DiCaprio, Kathleen M
Geisbert, Joan B
Ströher, Ute
Grolla, Allen
Bray, Mike
Fritz, Elizabeth A
Fernando, Lisa
Feldmann, Friederike
Hensley, Lisa E
Geisbert, Thomas W
PLoS Pathogens
Ebola viruses are highly lethal human pathogens that have received considerable attention in recent years due to an increasing re-emergence in Central Africa and a potential for use as a biological weapon. There is no vaccine or treatment licensed for human use. In the past, however, important advances have been made in developing preventive vaccines that are protective in animal models. In this regard, we showed that a single injection of a live-attenuated recombinant vesicular stomatitis virus vector expressing the Ebola virus glycoprotein completely protected rodents and nonhuman primates from lethal Ebola challenge. In contrast, progress in developing therapeutic interventions against Ebola virus infections has been much slower and there is clearly an urgent need to develop effective post-exposure strategies to respond to future outbreaks and acts of bioterrorism, as well as to treat laboratory exposures. Here we tested the efficacy of the vesicular stomatitis virus-based Ebola vaccine vector in post-exposure treatment in three relevant animal models. In the guinea pig and mouse models it was possible to protect 50% and 100% of the animals, respectively, following treatment as late as 24 h after lethal challenge. More important, four out of eight rhesus macaques were protected if treated 20 to 30 min following an otherwise uniformly lethal infection. Currently, this approach provides the most effective post-exposure treatment strategy for Ebola infections and is particularly suited for use in accidentally exposed individuals and in the control of secondary transmission during naturally occurring outbreaks or deliberate release.
Author Summary
Being highly pathogenic for humans and monkeys and the subject of former weapons programs makes Ebola virus one of the most feared pathogens worldwide today. Due to a lack of licensed pre- and post-exposure intervention, our current response depends on rapid diagnostics, proper isolation procedures, and supportive care of case patients. Consequently, the development of more specific countermeasures is of high priority for the preparedness of many nations. In this study, we investigated an attenuated vesicular stomatitis virus expressing the Ebola virus surface glycoprotein, which had previously demonstrated convincing efficacy as a vaccine against Ebola infections in rodents and monkeys, for its potential use in the treatment of an Ebola virus infection. Surprisingly, treatment of guinea pigs and mice as late as 24 h after lethal Ebola virus infection resulted in 50% and 100% survival, respectively. More important, 50% of rhesus macaques (4/8) were protected if treated 20 to 30 min after Ebola virus infection. Currently, this approach provides the most effective treatment strategy for Ebola infections and seems particularly suited for the use in accidental exposures and the control of human-to-human transmission during outbreaks.
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Protection against lethal challenge by Ebola virus-like particles produced in insect cells
Sun, Yuliang
Carrion, Ricardo
Ye, Ling
Wen, Zhiyuan
Ro, Young-Tae
Brasky, Kathleen
Ticer, Anysha E.
Schwegler, E. Ellen
Patterson, Jean L.
Compans, Richard W.
Yang, Chinglai
Ebola virus-like particles (VLPs) were produced in insect cells using a recombinant baculovirus expression system and their efficacy for protection against Ebola virus infection was investigated. Two immunizations with 50 ug Ebola VLPs (high dose) induced a high level of antibodies against Ebola GP that exhibited strong neutralizing activity against GP-mediated virus infection and conferred complete protection of vaccinated mice against lethal challenge by a high dose of mouse-adapted Ebola virus. In contrast, two immunizations with 10 ug Ebola VLPs (low dose) induced 5-fold lower levels of antibodies against GP and these mice were not protected against lethal Ebola virus challenge, similar to control mice that were immunized with 50 ug SIV Gag VLPs. However, the antibody response against GP were boosted significantly after a third immunization with 10 ug Ebola VLPs to similar levels as those induced by two immunizations with 50 ug Ebola VLPs, and vaccinated mice were also effectively protected against lethal Ebola virus challenge. Furthermore, serum viremia levels in protected mice were either below the level of detection or significantly lower compared to the viremia levels in control mice. These results show that effective protection can be achieved by immunization with Ebola VLPs produced in insect cells, which give high production yields, and lend further support to their development as an effective vaccine strategy against Ebola virus.
Related Articles
Vesicular Stomatitis Virus-Based Ebola Vaccine Is Well-Tolerated and Protects Immunocompromised Nonhuman Primates
Geisbert, Thomas W.
Daddario-DiCaprio, Kathleen M.
Lewis, Mark G.
Geisbert, Joan B.
Grolla, Allen
Leung, Anders
Paragas, Jason
Matthias, Lennox
Smith, Mark A.
Jones, Steven M.
Hensley, Lisa E.
Feldmann, Heinz
Jahrling, Peter B.
PLoS Pathogens
Ebola virus (EBOV) is a significant human pathogen that presents a public health concern as an emerging/re-emerging virus and as a potential biological weapon. Substantial progress has been made over the last decade in developing candidate preventive vaccines that can protect nonhuman primates against EBOV. Among these prospects, a vaccine based on recombinant vesicular stomatitis virus (VSV) is particularly robust, as it can also confer protection when administered as a postexposure treatment. A concern that has been raised regarding the replication-competent VSV vectors that express EBOV glycoproteins is how these vectors would be tolerated by individuals with altered or compromised immune systems such as patients infected with HIV. This is especially important as all EBOV outbreaks to date have occurred in areas of Central and Western Africa with high HIV incidence rates in the population. In order to address this concern, we evaluated the safety of the recombinant VSV vector expressing the Zaire ebolavirus glycoprotein (VSVΔG/ZEBOVGP) in six rhesus macaques infected with simian-human immunodeficiency virus (SHIV). All six animals showed no evidence of illness associated with the VSVΔG/ZEBOVGP vaccine, suggesting that this vaccine may be safe in immunocompromised populations. While one goal of the study was to evaluate the safety of the candidate vaccine platform, it was also of interest to determine if altered immune status would affect vaccine efficacy. The vaccine protected 4 of 6 SHIV-infected macaques from death following ZEBOV challenge. Evaluation of CD4+ T cells in all animals showed that the animals that succumbed to lethal ZEBOV challenge had the lowest CD4+ counts, suggesting that CD4+ T cells may play a role in mediating protection against ZEBOV.
Author Summary
Ebola virus is among the most lethal microbes known to man, with case fatality rates often exceeding 80%. Since its discovery in 1976, outbreaks have been sporadic and geographically restricted, primarily to areas of Central Africa. However, concern about the natural or unnatural introduction of Ebola outside of the endemic areas has dramatically increased both research interest and public awareness. A number of candidate vaccines have been developed to combat Ebola virus, and these vaccines have shown varying degrees of success in nonhuman primate models. Safety is a significant concern for any vaccine and in particular for vaccines that replicate in the host. Here, we evaluated the safety of our replication-competent vesicular stomatitus virus (VSV)-based Ebola vaccine in SHIV-infected rhesus monkeys. We found that the vaccine caused no evidence of overt illness in any of these immunocompromised animals. We also demonstrated that this vaccine partially protected the SHIV-infected monkeys against a lethal Ebola challenge and that there appears to be an association with levels of CD4+ lymphocytes and survival. Our study suggests that the VSV-based Ebola vaccine will be safe in immunocompromised populations and supports further study and development of this promising vaccine platform for its use in humans.
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Ebola GP-Specific Monoclonal Antibodies Protect Mice and Guinea Pigs from Lethal Ebola Virus Infection
Qiu, Xiangguo
Fernando, Lisa
Melito, P. Leno
Audet, Jonathan
Feldmann, Heinz
Kobinger, Gary
Alimonti, Judie B.
Jones, Steven M.
PLoS Neglected Tropical Diseases
Ebola virus (EBOV) causes acute hemorrhagic fever in humans and non-human primates with mortality rates up to 90%. So far there are no effective treatments available. This study evaluates the protective efficacy of 8 monoclonal antibodies (MAbs) against Ebola glycoprotein in mice and guinea pigs. Immunocompetent mice or guinea pigs were given MAbs i.p. in various doses individually or as pools of 3–4 MAbs to test their protection against a lethal challenge with mouse- or guinea pig-adapted EBOV. Each of the 8 MAbs (100 µg) protected mice from a lethal EBOV challenge when administered 1 day before or after challenge. Seven MAbs were effective 2 days post-infection (dpi), with 1 MAb demonstrating partial protection 3 dpi. In the guinea pigs each MAb showed partial protection at 1 dpi, however the mean time to death was significantly prolonged compared to the control group. Moreover, treatment with pools of 3–4 MAbs completely protected the majority of animals, while administration at 2–3 dpi achieved 50–100% protection. This data suggests that the MAbs generated are capable of protecting both animal species against lethal Ebola virus challenge. These results indicate that MAbs particularly when used as an oligoclonal set are a potential therapeutic for post-exposure treatment of EBOV infection.
Author Summary
Ebola virus (EBOV) causes acute hemorrhagic fever in humans and non-human primates with mortality rates up to 90%. So far there are no effective treatments available. This study evaluates the protective efficacy of 8 monoclonal antibodies (MAbs) against the Ebola virus surface glycoprotein, in mice and guinea pigs. Various combinations and doses of the neutralizing and non-neutralizing MAbs were tested, and a post-exposure treatment protocol was determined. There was 100% survival when guinea pigs received a mix of 3 neutralizing MAbs two days after a challenge with 1,000 LD50 of guinea pig-adapted EBOV. This data suggests that the MAbs generated are effective as a post-exposure therapeutic for a lethal Ebola virus infection. Development of a post-exposure therapeutic for an Ebola virus infection is vital due to the high lethality of the disease, the relative speed in which it kills, and the fact that no vaccine has been approved for human use. Additionally, is it unlikely that preventative vaccines will be employed, because Ebola virus infections occur primarily in Africa, and to date have only killed approximately 2,300 people making it financially unfeasible for a mass vaccination. Therefore, having an effective therapy in the event of an outbreak would be extremely beneficial.
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Primary pulmonary cytotoxic T lymphocytes induced by immunization with a vaccinia virus recombinant expressing influenza A virus nucleoprotein peptide do not protect mice against challenge.
Lawson, C M
Bennink, J R
Restifo, N P
Yewdell, J W
Murphy, B R
Journal of Virology
The nucleoprotein (NP) of influenza A virus is the dominant antigen recognized by influenza virus-specific cytotoxic T lymphocytes (CTLs), and adoptive transfer of NP-specific CTLs protects mice from influenza A virus infection. BALB/c mouse cells (H-2d) recognize a single Kd-restricted CTL epitope of NP consisting of amino acids 147 to 155. In the present study, mice were immunized with various vaccinia virus recombinant viruses to examine the effect of the induction of primary pulmonary CTLs on resistance to challenge with influenza A/Puerto Rico/8/34 virus. The minigene ESNP(147-155)-VAC construct, composed of a signal sequence from the adenovirus E3/19K glycoprotein (designated ES) and expressing the 9-amino-acid NP natural determinant (amino acids 147 to 155) preceded by an alanine residue, a similar minigene NP(Met 147-155)-VAC lacking ES, and a full-length NP-VAC recombinant of influenza virus were analyzed. The two minigene NP-VAC recombinants induced a greater primary pulmonary CTL response than the full-length NP-VAC recombinant. However, NP-specific CTLs induced by immunization with ESNP(147-155)-VAC did not decrease peak virus titer or accelerate clearance of virus in the lungs of mice challenged intranasally with A/PR/8/34. Furthermore, NP-specific CTLs induced by immunization did not protect mice challenged intranasally with a lethal dose of A/PR/8/34. Sequence analysis of the NP CTL epitope of A/PR/8/34 challenge virus obtained from lungs after 8 days of replication in ESNP(147-155)-VAC-immunized mice showed identity with that of the input virus, demonstrating that an escape mutant had not emerged during replication in vivo. Thus, in contrast to adoptively transferred CTLs, pulmonary NP-specific CTLs induced by recombinant vaccinia virus immunization do not have protective in vivo antiviral activity against influenza virus infection.
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Molecularly engineered vaccine which expresses an immunodominant T-cell epitope induces cytotoxic T lymphocytes that confer protection from lethal virus infection.
Klavinskis, L S
Whitton, J L
Oldstone, M B
Journal of Virology
Identification of a single viral T-cell epitope, associated with greater than 95% of the virus-specific cytotoxic T-lymphocyte (CTL) activity in BALB/c (H-2d) mice (J. L. Whitton, A. Tishon, H. Lewicki, J. Gebhard, T. Cook, M. Salvato, E. Joly, and M. B. A. Oldstone, J. Virol. 63:4303-4310, 1989), permitted us to design a CTL vaccine and test its ability to protect against a lethal virus challenge. Here we show that a single immunization with a recombinant vaccinia virus-lymphocytic choriomeningitis virus (LCMV) vaccine (VVNPaa1-201) expressing the immunodominant epitope completely protected H-2d mice from lethal infection with LCMV but did not protect H-2b mice. Furthermore, we show that the success or failure of immunization was determined entirely by the host class I major histocompatibility glycoproteins. The difference in outcome between mice of these two haplotypes was consistent with the presence or absence in the immunizing sequences of an epitope for CTL recognition and is correlated with the induction of LCMV-specific H-2-restricted CTL in H-2d mice. Protection is not conferred by a humoral immune response, since LCMV-specific antibodies were not detectable in sera from VVNPaa1-201-immunized mice. In addition, passive transfer of sera from vaccinated mice did not confer protection upon naive recipients challenged with LCMV. Hence, the molecular dissection of viral proteins can uncover immunodominant CTL epitope(s) that can be engineered into vaccines that elicit CTL. A single CTL epitope can protect against a lethal virus infection, but the efficacy of the vaccine varies in a major histocompatibility complex-dependent manner.
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Mechanisms of Immunity in Post-Exposure Vaccination against Ebola Virus Infection
Bradfute, Steven B.
Anthony, Scott M.
Stuthman, Kelly S.
Ayithan, Natarajan
Tailor, Prafullakumar
Shaia, Carl I.
Bray, Mike
Ozato, Keiko
Bavari, Sina
PLoS ONE
Ebolaviruses can cause severe hemorrhagic fever that is characterized by rapid viral replication, coagulopathy, inflammation, and high lethality rates. Although there is no clinically proven vaccine or treatment for Ebola virus infection, a virus-like particle (VLP) vaccine is effective in mice, guinea pigs, and non-human primates when given pre-infection. In this work, we report that VLPs protect Ebola virus-infected mice when given 24 hours post-infection. Analysis of cytokine expression in serum revealed a decrease in pro-inflammatory cytokine and chemokine levels in mice given VLPs post-exposure compared to infected, untreated mice. Using knockout mice, we show that VLP-mediated post-exposure protection requires perforin, B cells, macrophages, conventional dendritic cells (cDCs), and either CD4+ or CD8+ T cells. Protection was Ebola virus-specific, as marburgvirus VLPs did not protect Ebola virus-infected mice. Increased antibody production in VLP-treated mice correlated with protection, and macrophages were required for this increased production. However, NK cells, IFN-gamma, and TNF-alpha were not required for post-exposure-mediated protection. These data suggest that a non-replicating Ebola virus vaccine can provide post-exposure protection and that the mechanisms of immune protection in this setting require both increased antibody production and generation of cytotoxic T cells.
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Enhanced Protection against Ebola Virus Mediated by an Improved Adenovirus-Based Vaccine
Richardson, Jason S.
Yao, Michel K.
Tran, Kaylie N.
Croyle, Maria A.
Strong, James E.
Feldmann, Heinz
Kobinger, Gary P.
PLoS ONE
The Ebola virus is transmitted by direct contact with bodily fluids of infected individuals, eliciting death rates as high as 90% among infected humans. Currently, replication defective adenovirus-based Ebola vaccine is being studied in a phase I clinical trial. Another Ebola vaccine, based on an attenuated vesicular stomatitis virus has shown efficacy in post-exposure treatment of nonhuman primates to Ebola infection. In this report, we modified the common recombinant adenovirus serotype 5-based Ebola vaccine expressing the wild-type ZEBOV glycoprotein sequence from a CMV promoter (Ad-CMVZGP). The immune response elicited by this improved expression cassette vector (Ad-CAGoptZGP) and its ability to afford protection against lethal ZEBOV challenge in mice was compared to the standard Ad-CMVZGP vector.
Methodology/Principal Findings
Ad-CMVZGP was previously shown to protect mice, guinea pigs and nonhuman primates from an otherwise lethal challenge of Zaire ebolavirus. The antigenic expression cassette of this vector was improved through codon optimization, inclusion of a consensus Kozak sequence and reconfiguration of a CAG promoter (Ad-CAGoptZGP). Expression of GP from Ad-CAGoptZGP was substantially higher than from Ad-CMVZGP. Ad-CAGoptZGP significantly improved T and B cell responses at doses 10 to 100-fold lower than that needed with Ad-CMVZGP. Additionally, Ad-CAGoptZGP afforded full protections in mice against lethal challenge at a dose 100 times lower than the dose required for Ad-CMVZGP. Finally, Ad-CAGoptZGP induced full protection to mice when given 30 minutes post-challenge.
We describe an improved adenovirus-based Ebola vaccine capable of affording post-exposure protection against lethal challenge in mice. The molecular modifications of the new improved vaccine also translated in the induction of significantly enhanced immune responses and complete protection at a dose 100 times lower than with the previous generation adenovirus-based Ebola vaccine. Understanding and improving the molecular components of adenovirus-based vaccines can produce potent, optimized product, useful for vaccination and post-exposure therapy.
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Identification of Protective Epitopes on Ebola Virus Glycoprotein at the Single Amino Acid Level by Using Recombinant Vesicular Stomatitis Viruses
Takada, Ayato
Feldmann, Heinz
Stroeher, Ute
Bray, Mike
Watanabe, Shinji
Ito, Hiroshi
McGregor, Martha
Kawaoka, Yoshihiro
Journal of Virology
Ebola virus causes lethal hemorrhagic fever in humans, but currently there are no effective vaccines or antiviral compounds for this infectious disease. Passive transfer of monoclonal antibodies (MAbs) protects mice from lethal Ebola virus infection (J. A. Wilson, M. Hevey, R. Bakken, S. Guest, M. Bray, A. L. Schmaljohn, and M. K. Hart, Science 287:1664-1666, 2000). However, the epitopes responsible for neutralization have been only partially characterized because some of the MAbs do not recognize the short synthetic peptides used for epitope mapping. To identify the amino acids recognized by neutralizing and protective antibodies, we generated a recombinant vesicular stomatitis virus (VSV) containing the Ebola virus glycoprotein-encoding gene instead of the VSV G protein-encoding gene and used it to select escape variants by growing it in the presence of a MAb (133/3.16 or 226/8.1) that neutralizes the infectivity of the virus. All three variants selected by MAb 133/3.16 contained a single amino acid substitution at amino acid position 549 in the GP2 subunit. By contrast, MAb 226/8.1 selected three different variants containing substitutions at positions 134, 194, and 199 in the GP1 subunit, suggesting that this antibody recognized a conformational epitope. Passive transfer of each of these MAbs completely protected mice from a lethal Ebola virus infection. These data indicate that neutralizing antibody cocktails for passive prophylaxis and therapy of Ebola hemorrhagic fever can reduce the possibility of the emergence of antigenic variants in infected individuals.
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In silico prediction of Ebola Zaire GP1,2 immuno-dominant epitopes for the Balb/c mouse
Dutta, Debargh K.
Rhodes, Kelly
Wood, Steven C.
BMC Immunology
Ebola is a Filovirus (FV) that induces a highly communicable and deadly hemorrhagic fever. Currently, there are no approved vaccines to treat FV infections. Protection from FV infection requires cell mediated and humoral immunity. Glycoprotein GP1,2 Fc Zaire, a recombinant FV human Fc fusion protein, has been shown to confer protection against mouse adapted Zaire Ebola virus. The present studies are focused upon identifying immunodominant epitopes using in silico methods and developing tetramers as a diagnostic reagent to detect cell mediated immune responses to GP1,2 Fc.
The GP1,2 Ebola Zaire sequence from the 1976 outbreak was analyzed by both BIMAS and SYFPEITHI algorithms to identify potential immuno-dominant epitopes. Several peptides were synthesized and screened in flow-based MHC stability studies. Three candidate peptides, P8, P9 and P10, were identified and, following immunization in Balb/c mice, all three peptides induced IFN-γ as detected by ELISpot and intracellular staining.
Significantly, P8, P9 and P10 generated robust cytotoxic T-cell responses (CTL) as determined by a flow cytometry-based Caspase assay. Antigen specific cells were also detected, using tetramers. Both P9 and P10 have sequence homology with highly conserved regions of several strains of FV.
In sum, three immunodominant sequences of the Ebola GP1,2 have been identified using in silico methods that may confer protection against mouse adapted Ebola Zaire. The development of tetramer reagents will provide unique insight into the potency and durability of medical countermeasure vaccines for known bioterrorism threat agents in preclinical models.
Ebola; GP1,2; Immunodominant epitopes; CTL; Tetramer
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Demonstration of Cross-Protective Vaccine Immunity against an Emerging Pathogenic Ebolavirus Species
Hensley, Lisa E.
Mulangu, Sabue
Asiedu, Clement
Johnson, Joshua
Honko, Anna N.
Stanley, Daphne
Fabozzi, Giulia
Nichol, Stuart T.
Ksiazek, Thomas G.
Rollin, Pierre E.
Wahl-Jensen, Victoria
Bailey, Michael
Jahrling, Peter B.
Roederer, Mario
Koup, Richard A.
Sullivan, Nancy J.
PLoS Pathogens
A major challenge in developing vaccines for emerging pathogens is their continued evolution and ability to escape human immunity. Therefore, an important goal of vaccine research is to advance vaccine candidates with sufficient breadth to respond to new outbreaks of previously undetected viruses. Ebolavirus (EBOV) vaccines have demonstrated protection against EBOV infection in nonhuman primates (NHP) and show promise in human clinical trials but immune protection occurs only with vaccines whose antigens are matched to the infectious challenge species. A 2007 hemorrhagic fever outbreak in Uganda demonstrated the existence of a new EBOV species, Bundibugyo (BEBOV), that differed from viruses covered by current vaccine candidates by up to 43% in genome sequence. To address the question of whether cross-protective immunity can be generated against this novel species, cynomolgus macaques were immunized with DNA/rAd5 vaccines expressing ZEBOV and SEBOV glycoprotein (GP) prior to lethal challenge with BEBOV. Vaccinated subjects developed robust, antigen-specific humoral and cellular immune responses against the GP from ZEBOV as well as cellular immunity against BEBOV GP, and immunized macaques were uniformly protected against lethal challenge with BEBOV. This report provides the first demonstration of vaccine-induced protective immunity against challenge with a heterologous EBOV species, and shows that Ebola vaccines capable of eliciting potent cellular immunity may provide the best strategy for eliciting cross-protection against newly emerging heterologous EBOV species.
Author Summary
Ebola virus causes death, fear, and economic disruption during outbreaks. It is a concern worldwide as a natural pathogen and a bioterrorism agent, and has caused death to residents and tourists of Africa where the virus circulates. A vaccine strategy to protect against all circulating Ebola viruses is complicated by the fact that there are five different virus species, and individual vaccines provide protection only against those included in the vaccine. Making broad vaccines that contain multiple components is complicated, expensive, and poses challenges for regulatory approval. Therefore, in the present work, we examined whether a prime-boost immunization strategy with a vaccine targeted to one Ebola virus species could cross protect against a different species. We found that genetic immunization with vectors expressing the Ebola virus glycoprotein from Zaire blocked infection with a newly emerged virus species, Bundibugyo EBOV, not represented in the vaccine. Protection occurred in the absence of antibodies against the second species and was mediated instead by cellular immune responses. Therefore, single-component vaccines may be improved to protect against multiple Ebola viruses if they are designed to generate this type of immunity.
Related Articles
Immunization with a Single Major Histocompatibility Complex Class I-Restricted Cytotoxic T-Lymphocyte Recognition Epitope of Herpes Simplex Virus Type 2 Confers Protective Immunity
Blaney, Joseph E.
Nobusawa, Eri
Brehm, Michael A.
Bonneau, Robert H.
Mylin, Lawrence M.
Fu, Tong-Ming
Kawaoka, Yoshihiro
Tevethia, Satvir S.
Journal of Virology
We have evaluated the potential of conferring protective immunity to herpes simplex virus type 2 (HSV-2) by selectively inducing an HSV-specific CD8+ cytotoxic T-lymphocyte (CTL) response directed against a single major histocompatibility complex class I-restricted CTL recognition epitope. We generated a recombinant vaccinia virus (rVV-ES-gB498-505) which expresses the H-2Kb-restricted, HSV-1/2-cross-reactive CTL recognition epitope, HSV glycoprotein B residues 498 to 505 (SSIEFARL) (gB498-505), fused to the adenovirus type 5 E3/19K endoplasmic reticulum insertion sequence (ES). Mucosal immunization of C57BL/6 mice with this recombinant vaccinia virus induced both a primary CTL response in the draining lymph nodes and a splenic memory CTL response directed against HSV gB498-505. To determine the ability of the gB498-505-specific memory CTL response to provide protection from HSV infection, immunized mice were challenged with a lethal dose of HSV-2 strain 186 by the intranasal (i.n.) route. Development of the gB498-505-specific CTL response conferred resistance in 60 to 75% of mice challenged with a lethal dose of HSV-2 and significantly reduced the levels of infectious virus in the brains and trigeminal ganglia of challenged mice. Finally, i.n. immunization of C57BL/6 mice with either a recombinant influenza virus or a recombinant vaccinia virus expressing HSV gB498-505 without the ES was also demonstrated to induce an HSV-specific CTL response and provide protection from HSV infection. This finding confirms that the induction of an HSV-specific CTL response directed against a single epitope is sufficient for conferring protective immunity to HSV. Our findings support the role of CD8+ T cells in the control of HSV infection of the central nervous system and suggest the potential importance of eliciting HSV-specific mucosal CD8+ CTL in HSV vaccine design.
Related Articles
Role of Natural Killer Cells in Innate Protection against Lethal Ebola Virus Infection
Warfield, Kelly L.
Perkins, Jeremy G.
Swenson, Dana L.
Deal, Emily M.
Bosio, Catharine M.
Aman, M. Javad
Yokoyama, Wayne M.
Young, Howard A.
Bavari, Sina
The Journal of Experimental Medicine
Ebola virus is a highly lethal human pathogen and is rapidly driving many wild primate populations toward extinction. Several lines of evidence suggest that innate, nonspecific host factors are potentially critical for survival after Ebola virus infection. Here, we show that nonreplicating Ebola virus-like particles (VLPs), containing the glycoprotein (GP) and matrix protein virus protein (VP)40, administered 1–3 d before Ebola virus infection rapidly induced protective immunity. VLP injection enhanced the numbers of natural killer (NK) cells in lymphoid tissues. In contrast to live Ebola virus, VLP treatment of NK cells enhanced cytokine secretion and cytolytic activity against NK-sensitive targets. Unlike wild-type mice, treatment of NK-deficient or -depleted mice with VLPs had no protective effect against Ebola virus infection and NK cells treated with VLPs protected against Ebola virus infection when adoptively transferred to naive mice. The mechanism of NK cell–mediated protection clearly depended on perforin, but not interferon-γ secretion. Particles containing only VP40 were sufficient to induce NK cell responses and provide protection from infection in the absence of the viral GP. These findings revealed a decisive role for NK cells during lethal Ebola virus infection. This work should open new doors for better understanding of Ebola virus pathogenesis and direct the development of immunotherapeutics, which target the innate immune system, for treatment of Ebola virus infection.
virus-like particles; filoviruses; immunity; matrix protein; glycoprotein
Related Articles
Evaluation in Nonhuman Primates of Vaccines against Ebola Virus
Geisbert, Thomas W.
Pushko, Peter
Anderson, Kevin
Smith, Jonathan
Davis, Kelly J.
Jahrling, Peter B.
Emerging Infectious Diseases
Ebola virus (EBOV) causes acute hemorrhagic fever that is fatal in up to 90% of cases in both humans and nonhuman primates. No vaccines or treatments are available for human use. We evaluated the effects in nonhuman primates of vaccine strategies that had protected mice or guinea pigs from lethal EBOV infection. The following immunogens were used: RNA replicon particles derived from an attenuated strain of Venezuelan equine encephalitis virus (VEEV) expressing EBOV glycoprotein and nucleoprotein; recombinant Vaccinia virus expressing EBOV glycoprotein; liposomes containing lipid A and inactivated EBOV; and a concentrated, inactivated whole-virion preparation. None of these strategies successfully protected nonhuman primates from robust challenge with EBOV. The disease observed in primates differed from that in rodents, suggesting that rodent models of EBOV may not predict the efficacy of candidate vaccines in primates and that protection of primates may require different mechanisms.
Keywords: Ebola; macaque; vaccine; Vaccinia virus; replicon
Related Articles
Ebola virus glycoprotein Fc fusion protein confers protection against lethal challenge in vaccinated mice
Konduru, Krishnamurthy
Bradfute, Steven B.
Jacques, Jerome
Manangeeswaran, Mohanraj
Nakamura, Siham
Morshed, Sufi
Wood, Steven C.
Bavari, Sina
Kaplan, Gerardo G.
Ebola virus is a Filoviridae that causes hemorrhagic fever in humans and induces high morbidity and mortality rates. Filoviruses are classified as "Category A bioterrorism agents", and currently there are no licensed therapeutics or vaccines to treat and prevent infection. The Filovirus glycoprotein (GP) is sufficient to protect individuals against infection, and several vaccines based on GP are under development including recombinant adenovirus, parainfluenza virus, Venezuelan equine encephalitis virus, vesicular stomatitis virus (VSV) and virus-like particles. Here we describe the development of a GP Fc fusion protein as a vaccine candidate. We expressed the extracellular domain of the Zaire Ebola virus (ZEBOV) GP fused to the Fc fragment of human IgG1 (ZEBOVGP-Fc) in mammalian cells and showed that GP undergoes the complex furin cleavage and processing observed in the native membrane-bound GP. Mice immunized with ZEBOVGP-Fc developed T-cell immunity against ZEBOV GP and neutralizing antibodies against replication-competent VSV-G deleted recombinant VSV containing ZEBOV GP. The ZEBOVGP-Fc vaccinated mice were protected against challenge with a lethal dose of ZEBOV. These results show that vaccination with the ZEBOVGP-Fc fusion protein alone without the need of a viral vector or assembly into virus-like particles is sufficient to induce protective immunity against ZEBOV in mice. Our data suggested that Filovirus GP Fc fusion proteins could be developed as a simple, safe, efficacious, and cost effective vaccine against Filovirus infection for human use.
Related Articles
Crystal Structure of Swine Major Histocompatibility Complex Class I SLA-1*0401 and Identification of 2009 Pandemic Swine-Origin Influenza A H1N1 Virus Cytotoxic T Lymphocyte Epitope Peptides ▿
Zhang, Nianzhi
Qi, Jianxun
Feng, Sijia
Gao, Feng
Liu, Jun
Pan, Xiaocheng
Chen, Rong
Li, Qirun
Chen, Zhaosan
Li, Xiaoying
Xia, Chun
Gao, George F.
Journal of Virology
The presentation of viral epitopes to cytotoxic T lymphocytes (CTLs) by swine leukocyte antigen class I (SLA I) is crucial for swine immunity. To illustrate the structural basis of swine CTL epitope presentation, the first SLA crystal structures, SLA-1*0401, complexed with peptides derived from either 2009 pandemic H1N1 (pH1N1) swine-origin influenza A virus (S-OIVNW9; NSDTVGWSW) or Ebola virus (EbolaAY9; ATAAATEAY) were determined in this study. The overall peptide–SLA-1*0401 structures resemble, as expected, the general conformations of other structure-solved peptide major histocompatibility complexes (pMHC). The major distinction of SLA-1*0401 is that Arg156 has a "one-ballot veto" function in peptide binding, due to its flexible side chain. S-OIVNW9 and EbolaAY9 bind SLA-1*0401 with similar conformations but employ different water molecules to stabilize their binding. The side chain of P7 residues in both peptides is exposed, indicating that the epitopes are "featured" peptides presented by this SLA. Further analyses showed that SLA-1*0401 and human leukocyte antigen (HLA) class I HLA-A*0101 can present the same peptides, but in different conformations, demonstrating cross-species epitope presentation. CTL epitope peptides derived from 2009 pandemic S-OIV were screened and evaluated by the in vitro refolding method. Three peptides were identified as potential cross-species influenza virus (IV) CTL epitopes. The binding motif of SLA-1*0401 was proposed, and thermostabilities of key peptide–SLA-1*0401 complexes were analyzed by circular dichroism spectra. Our results not only provide the structural basis of peptide presentation by SLA I but also identify some IV CTL epitope peptides. These results will benefit both vaccine development and swine organ-based xenotransplantation.
Related Articles
A Single Sublingual Dose of an Adenovirus-Based Vaccine Protects against Lethal Ebola Challenge in Mice and Guinea Pigs
Choi, Jin Huk
Schafer, Stephen C.
Zhang, Lihong
Kobinger, Gary P.
Juelich, Terry
Freiberg, Alexander N.
Croyle, Maria A.
Molecular Pharmaceutics
Sublingual (SL) delivery, a non-invasive immunization method that bypasses the intestinal tract for direct entry into the circulation, was evaluated with an adenovirus (Ad5)-based vaccine for Ebola. Mice and Guinea pigs were immunized via the intramuscular (IM), nasal (IN), oral (PO) and SL routes. SL immunization elicited strong transgene expression in and attracted CD11c(+) antigen presenting cells to the mucosa. A SL dose of 1 × 108 infectious particles induced Ebola Zaire glycoprotein (ZGP)-specific IFN-γ+ T cells in spleen, bronchoalveolar lavage, mesenteric lymph nodes and submandibular lymph nodes (SMLN) of naïve mice in a manner similar to the same dose given IN. Ex vivo CFSE and in vivo cytotoxic T lymphocyte (CTL) assays confirmed that SL immunization elicits a notable population of effector memory CD8+ T cells and strong CTL responses in spleen and SMLN. SL immunization induced significant ZGP-specific Th1 and Th2 type responses unaffected by pre-existing immunity (PEI) that protected mice and Guinea pigs from lethal challenge. SL delivery protected more mice with PEI to Ad5 than IM injection. SL immunization also reduced systemic anti-Ad5 T and B cell responses in naïve mice and those with PEI, suggesting that secondary immunizations could be highly effective for both populations.
adenovirus 5; Ebola Zaire; sublingual; vaccine; mouse; Guinea pig; pre-existing immunity; CD4 T cell; memory response; toxicity
Related Articles
Neutralizing Antibody Fails to Impact the Course of Ebola Virus Infection in Monkeys
Oswald, Wendelien B
Geisbert, Thomas W
Davis, Kelly J
Geisbert, Joan B
Sullivan, Nancy J
Jahrling, Peter B
Parren, Paul W. H. I
Burton, Dennis R
PLoS Pathogens
Prophylaxis with high doses of neutralizing antibody typically offers protection against challenge with viruses producing acute infections. In this study, we have investigated the ability of the neutralizing human monoclonal antibody, KZ52, to protect against Ebola virus in rhesus macaques. This antibody was previously shown to fully protect guinea pigs from infection. Four rhesus macaques were given 50 mg/kg of neutralizing human monoclonal antibody KZ52 intravenously 1 d before challenge with 1,000 plaque-forming units of Ebola virus, followed by a second dose of 50 mg/kg antibody 4 d after challenge. A control animal was exposed to virus in the absence of antibody treatment. Passive transfer of the neutralizing human monoclonal antibody not only failed to protect macaques against challenge with Ebola virus but also had a minimal effect on the explosive viral replication following infection. We show that the inability of antibody to impact infection was not due to neutralization escape. It appears that Ebola virus has a mechanism of infection propagation in vivo in macaques that is uniquely insensitive even to high concentrations of neutralizing antibody.
Author Summary
Ebola virus is one of the most feared of human pathogens with a mortality that can approach 90% and an extremely rapid disease course that can lead to death within days of infection. Antibodies able to inhibit viral infection in culture, neutralizing antibodies, can typically prevent viral infection in animals and humans when present prior to infection, at sufficient concentration. Such neutralizing antibodies may be provided through passive administration or induced by vaccination. We have previously shown that a human neutralizing antibody can protect guinea pigs against Ebola virus. However, here we show that this antibody does not protect monkeys against Ebola virus and surprisingly appears to have very little impact upon the rapid course of infection, despite being present at very high levels in the blood of the monkeys. We conclude that administering antibody prior to or immediately following exposure to Ebola virus, for example, after an accident in a research setting or a bioterrorist attack, is unlikely to be effective in preventing disease. Recent successes in protecting monkeys against Ebola virus through vaccination may be independent of antibody, or, more likely, critically dependent on the cooperation of antibody and cellular immunity.
Related Articles
Successful Topical Respiratory Tract Immunization of Primates against Ebola Virus▿
Bukreyev, Alexander
Rollin, Pierre E.
Tate, Mallory K.
Yang, Lijuan
Zaki, Sherif R.
Shieh, Wun-Ju
Murphy, Brian R.
Collins, Peter L.
Sanchez, Anthony
Journal of Virology
Ebola virus causes outbreaks of severe viral hemorrhagic fever with high mortality in humans. The virus is highly contagious and can be transmitted by contact and by the aerosol route. These features make Ebola virus a potential weapon for bioterrorism and biological warfare. Therefore, a vaccine that induces both systemic and local immune responses in the respiratory tract would be highly beneficial. We evaluated a common pediatric respiratory pathogen, human parainfluenza virus type 3 (HPIV3), as a vaccine vector against Ebola virus. HPIV3 recombinants expressing the Ebola virus (Zaire species) surface glycoprotein (GP) alone or in combination with the nucleocapsid protein NP or with the cytokine adjuvant granulocyte-macrophage colony-stimulating factor were administered by the respiratory route to rhesus monkeys—in which HPIV3 infection is mild and asymptomatic—and were evaluated for immunogenicity and protective efficacy against a highly lethal intraperitoneal challenge with Ebola virus. A single immunization with any construct expressing GP was moderately immunogenic against Ebola virus and protected 88% of the animals against severe hemorrhagic fever and death caused by Ebola virus. Two doses were highly immunogenic, and all of the animals survived challenge and were free of signs of disease and of detectable Ebola virus challenge virus. These data illustrate the feasibility of immunization via the respiratory tract against the hemorrhagic fever caused by Ebola virus. To our knowledge, this is the first study in which topical immunization through respiratory tract achieved prevention of a viral hemorrhagic fever infection in a primate model.
Related Articles
A Single Dose Respiratory Recombinant Adenovirus-Based Vaccine Provides Long-Term Protection for Non-Human Primates from Lethal Ebola Infection
Choi, Jin Huk
Jonsson-Schmunk, Kristina
Qiu, Xiangguo
Shedlock, Devon J.
Strong, Jim
Xu, Jason X.
Michie, Kelly L.
Audet, Jonathan
Fernando, Lisa
Myers, Mark J.
Weiner, David
Bajrovic, Irnela
Tran, Lilian Q.
Wong, Gary
Bello, Alexander
Kobinger, Gary P.
Schafer, Stephen C.
Croyle, Maria A.
Molecular Pharmaceutics
As the Ebola outbreak in West Africa continues and cases appear in the United States and other countries, the need for long-lasting vaccines to preserve global health is imminent. Here, we evaluate the long-term efficacy of a respiratory and sublingual (SL) adenovirus-based vaccine in non-human primates in two phases. In the first, a single respiratory dose of 1.4 × 109 infectious virus particles (ivp)/kg of Ad-CAGoptZGP induced strong Ebola glycoprotein (GP) specific CD8+ and CD4+ T cell responses and Ebola GP-specific antibodies in systemic and mucosal compartments and was partially (67%) protective from challenge 62 days after immunization. The same dose given by the SL route induced Ebola GP-specific CD8+ T cell responses similar to that of intramuscular (IM) injection, however, the Ebola GP-specific antibody response was low. All primates succumbed to infection. Three primates were then given the vaccine in a formulation that improved the immune response to Ebola in rodents. Three primates were immunized with 2.0 × 1010 ivp/kg of vaccine by the SL route. Diverse populations of polyfunctional Ebola GP-specific CD4+ and CD8+ T cells and significant anti-Ebola GP antibodies were present in samples collected 150 days after respiratory immunization. The formulated vaccine was fully protective against challenge 21 weeks after immunization. While diverse populations of Ebola GP-specific CD4+ T cells were produced after SL immunization, antibodies were not neutralizing and the vaccine was unprotective. To our knowledge, this is the first time that durable protection from a single dose respiratory adenovirus-based Ebola vaccine has been demonstrated in primates.
adenovirus; Ebola; non-human primate; vaccine; respiratory; sublingual
Related Articles
Protective mAbs and Cross-Reactive mAbs Raised by Immunization with Engineered Marburg Virus GPs
Fusco, Marnie L.
Hashiguchi, Takao
Cassan, Robyn
Biggins, Julia E.
Murin, Charles D.
Warfield, Kelly L.
Li, Sheng
Holtsberg, Frederick W.
Shulenin, Sergey
Vu, Hong
Olinger, Gene G.
Kim, Do H.
Whaley, Kevin J.
Zeitlin, Larry
Ward, Andrew B.
Nykiforuk, Cory
Aman, M. Javad
Berry, Jody
Saphire, Erica Ollmann
PLoS Pathogens
The filoviruses, which include the marburg- and ebolaviruses, have caused multiple outbreaks among humans this decade. Antibodies against the filovirus surface glycoprotein (GP) have been shown to provide life-saving therapy in nonhuman primates, but such antibodies are generally virus-specific. Many monoclonal antibodies (mAbs) have been described against Ebola virus. In contrast, relatively few have been described against Marburg virus. Here we present ten mAbs elicited by immunization of mice using recombinant mucin-deleted GPs from different Marburg virus (MARV) strains. Surprisingly, two of the mAbs raised against MARV GP also cross-react with the mucin-deleted GP cores of all tested ebolaviruses (Ebola, Sudan, Bundibugyo, Reston), but these epitopes are masked differently by the mucin-like domains themselves. The most efficacious mAbs in this panel were found to recognize a novel "wing" feature on the GP2 subunit that is unique to Marburg and does not exist in Ebola. Two of these anti-wing antibodies confer 90 and 100% protection, respectively, one hour post-exposure in mice challenged with MARV.
Author Summary
The filoviruses have caused multiple outbreaks among humans this decade, including a 90% lethal outbreak of Marburg virus in Angola and a significant, sustained outbreak of Ebola virus in West Africa. The viral surface glycoprotein (GP), which enables filoviruses to infect host cells, is the primary target of the immune system. Antibodies that target filovirus GP have been shown to provide life-saving therapy in nonhuman primates. However, the majority of known antibodies are only reactive against Ebola virus and not other emerging filoviruses. In this study, we present ten antibodies against Marburg virus, elicited by immunization of mice using engineered forms of its GP. Surprisingly, two antibodies exhibit some cross-reactivity to ebolaviruses (including species Ebola, Sudan, Bundibugyo, Reston). Other antibodies in this panel recognize a novel "wing" feature on a portion of GP that is unique to Marburg and does not exist in ebolaviruses, and protect 90%-100% of mice from lethal exposure. These antibodies, and their structural and functional analysis presented here, illuminate directions forward for therapeutics against Marburg virus.
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Pioneer Bank picks new VP of commercial debt TROY — Pioneer Bank has named Dave Blessing its new vice president of commercial structured debt. In this role, Blessing will be responsible for commercial structured finance programs for the bank, managing lending partnerships within the Capital Region and identifying opportunities to generate non-traditional lending opportunities in addition to ongoing relationship management. Blessing was previously vice president of commercial services at the bank, where he assisted in ...
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The past two weeks have been quite busy in relation to my forthcoming book Coffeehouse Theology: Reflecting on God in Everyday Life with NavPress this coming September. I thought that my work would basically be done when I finished writing the book. However, that appears to only have been half the job.
I have now entered the publicity season, and the first event on that calendar is finding a list of endorsers.
I began working on this by making sure I had plenty of information available about the book project. If you click on the Coffeehouse Theology link at the top of this web site, you'll see that I've revamped the page: inserted a better introduction to the book, pasted in some advance praise I received, and stuck a relatively up-to-date summary of each chapter. The "punathon" remains at the bottom of the page.
After that I spent an entire day writing to various Christian writers and leaders who may be interested in my project. The following two weeks were filled with all kinds of e-mails and research to dig up addresses and to follow up in various ways. I really had no idea how involved this can be!
So far I'm really happy with the list of Christian writers and ministry leaders who are willing to give my work a look. Of course I can't mention specific names until things are settled and in place.
I'm not 100% sure about my next step in the publicity drive since I still have quite a bit of time, but for the time being I'm going to be working on developing a few ways I can improve the buzz for the book. Once again this is uncharted waters, but I've spent enough time looking at what other authors have done that I have a few ideas jotted down. We'll see what happens next.


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Merridee's Apple Brownies With Butter Sauce
Total Time
Prep 15 mins
Cook 35 mins
I haven't tried these yet but they look yummy. Recipe from Merridee's cookbook out of Franklin, TN.
Ingredients Nutrition
For Brownies
- 1⁄4 cup shortening
- 1 cup sugar
- 1 egg
- 1⁄2 teaspoon cinnamon
- 1⁄2 teaspoon nutmeg
- 1 cup flour
- 1 teaspoon salt
- 1 teaspoon baking soda
- 2 cups tart apples (thinly sliced)
- 1⁄2 cup walnuts (chopped)
Butter sauce
- 1⁄2 cup butter
- 1 teaspoon vanilla
- 1⁄2 cup milk
- 1⁄2 cup sugar
- 1⁄2 cup brown sugar
- Preheat oven to 350 degrees.
- For brownies cream shortening, sugar and egg for one minute.
- Add sifted dry ingredients and blend thoroughly.
- Stir in apples and walnuts.
- Pour into greased and floured pie pan.
- Bake for 30 minutes or until golden brown.
- For butter sauce combine all ingredients except vanilla and bring to a rolling boil.
- Remove from heat and add vanilla.
- Serve over warm brownies.
Most Helpful
The brownies were yummy, but the sauce was a bit too sweet.
These turned out very very good - a good way to use up soft apples. The only problem I had is the butter sauce got curdled with the milk. I ended up straining it and using it anyway. Tastes great!


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Self-Made Man
One Woman's Year Disguised as a Man
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About the authorNorah Vincent is the author of the New York Times bestseller Self-Made Man. Previously, she wrote a nationally syndicated op-ed column for the Los Angeles Times. Her work has also appeared in The New York Times, The New Republic, The Village Voice, and The Washington Post. She lives in New York City.
moreA journalist's provocative and spellbinding account of her eighteen months spent disguised as a man.
Norah Vincent became an instant media sensation with the publication of Self-Made Man, her take on just how hard it is to be a man, even in a man's world. Following in the tradition of John Howard Griffin (Black Like Me), Vincent spent a year and a half disguised as her male alter ego, Ned, exploring what men are like when women aren't around. As Ned, she joined a bowling team, took a high-octane sales job, went on dates with women (and men), visited strip clubs, and even managed to infiltrate a monastery and a men's therapy group. At once thought-provoking and pure fun to read, Self-Made Man is a sympathetic and thrilling tour de force of immersion journalism.
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Penguin Publishing Group; January 2006
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Title: Self-Made Man
Author: Norah Vincent
Read online, or download in secure EPUB
Title: Self-Made Man
Author: Norah Vincent
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Category Archive for: 'Winter'
Roasted Root Vegetables with Rosemary & Honey
Root vegetables in the winter are not dainty. The produce that is harvested at this time of year is big and gnarled, but hopefully, it has been sweetened by frost. The roots have put on some serious bulk to last the season and when they are plucked from the soil they look like beasts. They sure don't taste like monsters (or …Read More
Evergreen Pressed Evergreen Herb Candles
These DIY pressed herb candles are perfect for anytime of year because they are created with evergreen herbs which are always available. Using beeswax or soy wax, canning jars, evergreen clippings, and essential oils, these candles make for beautiful handmade gifts and home decor that brings the outdoors in. Since I began working at a local apothecary here in Portland, Oregon, …Read More
How to Care for a Fresh Christmas Tree
Your perfect Christmas tree is only perfect if it lasts through the season! With these tips, you will be able to choose a tree that is fresh as well as learn how to keep it beautiful throughout the holidays. Be sure to also check out the recycling ideas at the end of this post. Head out to a local tree …Read More
Check Out These Cool Characters! The Top Plants for Winter Garden Interest
Winter doesn't have to be white in the garden. Even if there is a blanket of snow covering the soil, these colorful characters will add some pizzazz to the winter garden with their showy limbs, bright berries, and even some flowers! Here are some ideas for what to plant for winter garden interest. Emerald Colonnade Holly This beautiful evergreen …Read More
Season's Greetings! A Kitchy Printable Herb Packet Gift Idea
Quick and easy holiday gifts are essential for surviving the holiday season. This free printable "Season's Greetings" Herb Packet is perfect for small gifts and can easily fit into the mail with your holiday greeting cards! Use herbs harvested from your own garden to create flavorful seasoning mixes paired with a simple and delicious recipe. Materials: Cardstock Ruler & X-acto …Read More
Gardening For Your Front Door: Making a Fresh Wreath
Every year we like to hang a different wreath on our front door during the holidays. We've amassed a collection of wreaths to match the holiday season trends of sparkly red berries, Christmas ornaments, peacock feathers, and square-shaped boxwood but my favorites are most certainly natural, fresh wreaths. A fresh wreath makes for an individual work of art that smells …Read More


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Pats Just Going to Take It from Joey Porter
Is it even news anymore that Joey Porter runs his mouth? It's like, "I saw some birds fly, the grass is green, oxygen passes through our lungs, Joey Porter is trash-talking his opponents." It's only news if those things stop happening, or in Porter's case, the object of his derision replies in a noteworthy way.
This week, Porter was bringing up a game from last season when he thought the Pats unnecessarily ran up the score, before issuing the warning: "Now they've got to come into our yard. All the little antics they had before the game and stuff like that, they're not going to try that in our house."
Of Course the Dolphins miraculously ended the Patriots' incredible regular-season win streak during week three, 38-13. The rematch is set for this weekend at Dolphin Stadium, and the Patriots' reaction to Porter's mouth is to put their tails between their legs and basically agree.
"When you have 12 and a half, 13 and a half sacks, or whatever he's got in 10 games, you can pretty much say what you want in this league and get away with it," Patriots fullback Heath Evans responded Tuesday. "Until we do something about him, he's earned the right to say what he wants to say. They're 1-0 against us this year."
--Kyle Munzenriederrant
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First Look at Dong Chun Hong, Bringing Seoul-Based Korean-Chinese to K-Town
RSVP for a chance to attend the grand opening party.By Jenny Miller
RSVP for a chance to attend the grand opening party.By Jenny Miller
The offerings, which might be better suited to vegetarians, earn a solid B from lunch authority Zach Brooks.By Hadley Tomicki
It beats scratching it out with soccer moms over the last Tickle Me Elmo.By Hadley Tomicki
And so far, it's the best thing on the menu.By Hadley Tomicki
Mystery meat is increasingly being replaced by sushi and pork ramen.
Or maybe UrbanSpoon already pinpointed the location and it's game over.By Hadley Tomicki
The Korean sandwich shop stands a much better chance of survival with the SMC kids.By Hadley Tomicki
"Every kitchen that I'm at, any kitchen, if there's something coming off the stove, you can bet my spoon's coming out of that."By Lesley Balla
Asian and Italian restaurants are proving prominent on fall's bill of fare.By Jenny Miller
The Korean restaurant has been gutted.By Jenny Miller
The L.A. Weekly critic also gets initiated into the cult of Mother Dough Pizza.By Hadley Tomicki
The restaurant wraps ssam in bao for a creative take on lunch.By Hadley Tomicki
The lawsuit centers on an incident at Justin's, the rap mogul's Atlanta restaurant.
Judges include Heroes actor James Kyson and blogger Sarah Gim.By Hadley Tomicki
Will the city finally get a reliable source for the tacos that's not a truck?By Jenny Miller
Already, the television toque is arguing about an opening date with partner Darren Dzienciol.By Hadley Tomicki
The restaurant is bracing for possible change in six months, following news of a new ownership.By Hadley Tomicki
Incredible things are happening here, while the restaurant does its best to shake off the ghosts of Gyenari.By Hadley Tomicki
The once-lackluster street has seen a food revolution of its own with the introduction of Roy Choi's rice bowls and Matt Kang's coffee and ice cream shop.By Hadley Tomicki
"Korean cuisine is a 2 trick donkey," writes the controversial blogger, not afraid to stir up another fight.By Hadley Tomicki


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Related quotes
"...drought conditions throughout the Greater Horn of Africa are likely to continue in most of the areas between February until at least the beginning of April."
"We're still in a long-term drought, but short-term-wise, yes, the drought conditions have been eased. You can tell everything is greening up, and the fire threat is becoming very low."
"We were definitely in drought conditions. It's nice to get little shots of precipitation instead of one big gulp, which we really don't need. This will really help the farmers out."


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NOTE: Do not read on if you have not seen Season 3, Episode 9 of FX's The Americans, titled "Do Mail Robots Dream of Electric Sheep?"
People are not being polite and things are getting really real as we head into the season finale build-up.
Elizabeth spills all of her beans to an old lady she has to kill. Hans murders Todd. Philip grimaces at Gabriel because he's mad at how he's helping Elizabeth turn Paige. Stan and Oleg are oh-so-close to jail-breaking Nina.
And Martha. Martha knows all about Clark, now. The first scene last night was the heaviest one I've ever watched. Is she going to snap? What about that mail robot?
Here's what you all thought:
"@UD_j1mb0: Martha wants to "turn the page" and Elizabeth wants to "turn the Paige" #TheAmericans" this is awesome!
— Christy Roach (@christyintx) March 26, 2015
Philip is realizing that he, like most of us, has severely underestimated the steel that Martha is made of. #TheAmericans
— Chelle G (@MNInsomniac) March 26, 2015
Philip trusts Martha more than Elizabeth in some ways. At least in terms of acting w/out consultation. #TheAmericans
— Jim Naughton (@JimNaught) March 26, 2015
#TheAmericans Martha thinking about the mail robot like pic.twitter.com/goMIchwsFJ
— lolli (@lollimadness) March 26, 2015
Martha'll protect "Clarke," Phillip's on his own #TheAmericans
— Tara Swanson (@tvswan) March 26, 2015
Let's not forget... Martha has a gun. #TheAmericans
— Lauren Bradshaw (@flickchickdc) March 26, 2015
And then, this might be the best pitch for a television show, ever:
I really want a spinoff of #TheAmericans that's a wacky workplace comedy about the rest of the staff of the travel agency.
— Adam Davis (@amdhit) March 26, 2015
Is Martha long for this world? Tweet me @karenfratti
The Americans airs Wednesdays at 10 p.m. ET on FX.
More:The Americans The Americans Fx The Americans Fx Recap The Americans Recap The Americans Season 3
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Home › Forums › General Karting Discussion › Trustworthy shop in west michigan
This topic contains 11 replies, has 5 voices, and was last updated by Ryan Ouellette 4 months, 1 week ago.
June 5, 2016 at 3:45 pm #66824
Hi yall. Im about to lay down some cash and buy myself a TaG kart. dont really want to buy it online because there are too many scammers out. Anybody here know any trustworthy shop or any owner near west michigan that can help me find a used good condition kart?June 5, 2016 at 4:06 pm #66825
Ryan OuelletteParticipant
I'm not near Michigan but I have a great used CRG with a Rotax available…..June 5, 2016 at 4:09 pm #66826
what year and how used? price?June 5, 2016 at 4:25 pm #66827
Walt GiffordParticipant
I've never bought a used kart that didn't need allot of work to bring it up to aircraft standards. It's not that people are scammers they're just not the best mechanics. Get pics of the bottom. If the frame rails are good everything else can be fixed.
FAA certified jet engine and aircraft technician,
Nicholson Speedway class champion 2001,
Yamaha KT100 Service Center,
41 years karting experience
June 6, 2016 at 7:55 am #66857
- This reply was modified 4 months, 2 weeks ago by Walt Gifford.
Dennis ChappellParticipant
Google MRP Kart shop Tell Steve Dennis sent ya. Three Oaks Mi. west side of state.
June 7, 2016 at 6:20 pm #66934
- This reply was modified 4 months, 2 weeks ago by Dennis Chappell.
formosakid, where are you located, I'm in Grand Rapids. Are you running sprint tracks or the road racing. If you need some help I'm in my 43rd year karting may I can help or point you in the right direction.June 9, 2016 at 5:59 pm #67070
Bob Cole, Im in grand rapids area as well.June 10, 2016 at 7:09 pm #67118
Where are you looking to race, Ravenna, Lansing or any one of our four big tracks. We just was at Ravenna with a 206 against mod clones, my kid won. Next weekend I go to Mid Ohio to run my leopard.We ran at MIS a couple weeks ago. So what's your direction?June 13, 2016 at 6:34 am #67174
Dennis ChappellParticipant
Bob is Mr. Karter he will send you in the right direction. Your in good hands.June 13, 2016 at 12:26 pm #67194
just trying to get a good TaG or Lo206 kart. What four track we got here in west michigan. so far i only know about ravennaJune 13, 2016 at 6:20 pm #67201
Formosa Kid I sent you a private message , call me, even in the day time I'm retired BOBJune 14, 2016 at 6:40 am #67220
Ryan OuelletteParticipant
I have 2 CRG's. Both are in great condition. 2008 Road Rebel and 2010 Blackstar. The chassis are used but no flat spotting. I run them on a regular basis in my local club. They were stripped down, cleaned up and put back together this past winter. The Blackstar has not been used this year. The Road Rebel has been used in 3 events. I set pole this past weekend with a 3rd in pre final and 2nd in final. I can assure you the chassis' need nothing. I also have a Rotax that can be mounted to either chassis. It is a 2010 with less than 15 hours since new. No rebuilds. It was an engine from SSC East , from the Rotax Nationals. Great package. Let me know. Email to email@example.com
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This is going back a decade or more ago I believe, but....
...there was once an emergency landing made on the offramp of the Union Turnpike Exit on the Clearview Expressway in Queens, New York.
This took place in either the late 80's or the early 90's.
It was a small tiny aircraft that just needed to land. No one was hurt, and I believe there were only one or two passengers onboard.
Can anyone link me to an article, refresh my memory of exactly what happened, or better yet (oh boy!) show me a picture of it?
This would be me a very happy boy.
Thank you in advance.


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... ad from what is now Zimbabwe and established numerous ruling houses. These came in conflict with the Transvaal republic in the latter half of the 19th century resulting in a campaign against Chief Mphephu by the Transvaal government. The chief was defeated and the Venda area was annexed in 1898. Venda was a distinct administrative unit within South Africa before it became officially...


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Skip to comments.Rasmussen: NV O 50% / R 48% (Inds: R 59% / O 37%!!!)
Posted on 10/24/2012 5:07:33 PM PDT by sf4dubya
President Obama still receives 50% of the vote in Nevadas tight presidential race.
The latest Rasmussen Reports telephone survey of Likely Nevada voters, taken the night after the final presidential debate, shows Obama with 50% support to Mitt Romneys 48%. One percent (1%) prefers some other candidate, and one percent (1%) is undecided.
Among voters in Nevada not affiliated with either major political party, Romney leads Obama 59% to 37%.
(Excerpt) Read more at rasmussenreports.com ...
Looks like even Rasmussen is grossly oversampling Democrats/undersampling Republicans and independents. I wonder what his response rate is now. Compared to the other pollsters that report shrinking rates, now well below 10%.
Rasmussen must have called a few graveyards in addition to the houses. We all know how the dead vote.
We should not pick and choose what Ras poll we like. Scary times
I think people are missing the news... 22 pt lead for Romney among Independents in Nevada is huge.
The independents are nowhere near participating in early voting here as much as the GOP/Dems.
Nevada has a huge Mormon population that is being completely ignored .
Reid wins because he wins that voter block !
Just watch
Our response to unfavorable polls is getting a little too knee-jerk dismissive. This one should be taken a little more seriously.
This tells me it's very very close in Nevada. With the unions, Obama probably has a slight advantage. More work needs to be done in Nevada.
I think the reaction was due to the 22 point lead among indies. How can Romney lose IF the lead is really that big?
Amen! Although I'm continually perplexed at EVERY poll showing Romney up huge with Independents, who we've been told always decide elections, and still barely breaking even. Don't get it.
Office of Nevada Secretary of State Ross Miller
2012 General Election
Week 1 Early Voting Turnout
Updated: 11:45 am 10/24/2012
Dem 47.30%
Rep 35.92%
Other 16.78%
Absent/Mail-In Ballots
Dem 42.29%
Rep 42.16%
Other 15.55%
I agree. One of the things I would like to better understand though is what demographics are going for Romney and which states those favor him. Romney seems to be over-performing with whites. How about with Latinos? How do those populations compare in NV vs CO. Why is Romney ahead (hopefully) in CO but not in NV?
These numbers assume that all the democrats returning absentee ballots are actually voting for Obama. Do we know that is the case?
Those numbers I posted was for turnout so people could see how much in Clark and Washoe is still remaining to vote. If you go by Rasmussen, anywhere between 5-10% Dems could vote for Romney. We won't know until Election Day, obviously.
This whole thing in Nevada is to get all the independents and Republicans out to vote to even dent what is going to happen in Clark county (Las Vegas) with the SEIU bussing their members to the polls.
Totally impossible for Obama to win Nevada with that Indy number.
Totally impossible for Obama to win Nevada with that Indy number.
You would be correct if the rule of law was observed in Nevada elections. Don't discount voter fraud FRiend. It's real in large cities and it impacts our nation in a huge way. Just examine the Al Franken margin of victory and what has been discovered since that election. Erase that fraud and we don't have Obamacare?
The busier the person, the less likely to respond to a poll; Without a representative sample the poll will not reflect the likely vote. The push polls would not care because they are designed for propaganda. For the campaign polls the professional pollsters and their customers care very much that the result be close to the likely outcome.
Top number is concerning, especially since the Kenyan is at 50%.
Internals look great though.
Republican SOS elected in 2010 in CO and a Dem SOS in NV.
7% higher Latino population in NV, and about 4% higher black population in NV as compared to CO.
My guess.
1. Latino Population, State of Nevada
CO: 4.3% black; 20.9% hispanic
My guess.
1. Latino Population, State of Nevada
Rasmussen is slitting his own throat to satisfy leftists who dumped millions into his bank account.
Yeah. Nevada is likely gone as a GOP state. Too many illegals.
We need to win this back in Ohio, Iowa and Wisconsin.
NV went for Obama over McCain 55-43 in 2008.
I am afraid so. Plus we have a Democrat secretary of state who isn't interested in ensuring the integrity of our elections, plus no mechanism in place whatsoever that would prevent illegal aliens from voting. It is an honor system with no repercussions for violations. Sure there is a threat, but that is all there is.
Obama won Nevada by 13 percentage points in 2008 and now only leads by 2, big big swing away from the anointed one.
Yes, that is all true. But we have to figure in the vast amount of fraud that will be perpetrated by the Democrats and their machine. In 2010 Sharon Angle was ahead in the polls against Harry Reid. I don't think he missed a moment of sleep worrying about the election. After his people did their thing, he "won".
Why do the Democrats always seem to do so much better at early voting? Do they make a point to drag people to the polls...whether one agrees with early voting, the rules are the rules, and the GOP should play be them and bus people to the polls too.
Is there some reason I'm missing?
This is the key stat.
This is why they are forced to oversample Democrats.
BUT no one can quantify Nevada's 7% Mormon pop. Yet.
Buses full of illegals from the casino kitchens and the chambermaids.
Although, it's worth noting registration is closed in Nevada.
I've listened to a couple of programs now about Nevada, and they all say that Romney's problem in Nevada is the Hispanic vote.
He somehow needs to shake loose some more Hispanic voters or increase the ground game on the supporters that he does have.
Nevada is critical to any plan he has to win without winning Ohio.
Geez tough enough that his hoard from this south has about ruined our country. Now they get to finish us off.
any Ohio news
PMSNBC is saying (all know) Ohio is inevitable to Obama via 2 to 1 early votes for the commander in fraud.
The hoard from the south is one issue, and those Hispanics who are lifelong US citizens for generations is another issue.
Romney is losing the 2nd group, and it really baffles me. Demographically speaking, Hispanic Americans should be more Catholic and evangelical, and therefore, more concerned about government encroachment on religious liberty, particularly on issues of life. Romney has made ObamaCare an element of his campaign, but he's not focused on the Catholic/evangelical alarm that the government is telling them how to handle their religious beliefs.
This would involve dealing with "abortion and contraception" and Romney considers that to be a 3rd rail.
He needs a good ad that simply addresses government telling churches what to believe. That might shake loose a few more Hispanic voters. JMHO, and I might well be wrong, but it's a gut feel.
I was encouraged the other day that Romney had counseled a woman not to receive an abortion when her doctor was telling her otherwise. That was one of the cases that early reports said Gloria Allred might be taking up to throw in Romney's face. Turns out it wasn't the one, but it did give some hope that Romney might be more concerned with life than other information had indicated. It was the first time I'd heard this story.
That's because his claims to conservatism are motivated by necessity rather than genuine values.
I tend to agree with you, but, like I said, he apparently did disagree pretty strongly with medical folks who were telling a woman in his parish — he was bishop — to receive an abortion due to some complication.
I don't know when this was, his age, the result, etc. I just know that the woman was one candidate for Gloria Allred to use to bash him. So, apparently, the woman held it against him. That makes me wonder if she's upset that he told her not to get the abortion or upset that she followed his advice. I don't really know.
re: Although, it's worth noting registration is closed in Nevada.
True, but they can still vote for the dead. And without a doubt, most of those illegals from the casino kitchens and chambermaids were registered before the deadline.
Take that PPP!
Dueling polls out of Nevada. The key here is that Nevada is now mustwin for Obama but not for Romney, now that Colorado is looking good.
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