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- Osteogenic Cell Cultures Cannot Utilize Exogenous Sources of Synthetic Polyphosphate for Mineralization. [JOURNAL ARTICLE]
- J Cell Biochem 2014 Jul 16.
Phosphate is critical for mineralization and deficiencies in the regulation of free phosphate lead to disease. Inorganic polyphosphates (polyPs) may represent a physiological source of phosphate because they can be hydrolyzed by biological phosphatases. To investigate whether exogenous polyP could be utilized for mineral formation, mineralization was evaluated in two osteogenic cell lines, Saos-2 and MC3T3, expressing different levels of tissue non-specific alkaline phosphatase (tnALP). The role of tnALP was further explored by lentiviral-mediated overexpression in MC3T3 cells. When cells were cultured in the presence of three different phosphate sources, there was a strong mineralization response with β-glycerophosphate (βGP) and orthophosphate (Pi) but none of the cultures sustained mineralization in the presence of polyP (neither chain length 17-Pi nor 42-Pi). Even in the presence of mineralizing levels of phosphate, low concentrations of polyP (50 μM) were sufficient to inhibit mineral formation. EDS confirmed the presence of apatite-like mineral deposits in MC3T3 cultures supplemented with βGP, but not in those with polyP. While von Kossa staining was consistent with the presence or absence of mineral, an unusual Alizarin staining was obtained in polyP-treated MC3T3 cultures. This staining pattern combined with low Ca:P ratios suggests the persistence of Ca-polyP complexes, even with high residual ALP activity. In conclusion, under standard culture conditions, exogenous polyP does not promote mineral deposition. This is not due to a lack of active ALP, and unless conditions that favor significant processing of polyP are achieved, its mineral inhibitory capacity predominates. J. Cell. Biochem. © 2014 Wiley Periodicals, Inc.
- Potent Neutralization of Vaccinia Virus by Divergent Murine Antibodies Targeting a Common Site of Vulnerability in L1 Protein. [JOURNAL ARTICLE]
- J Virol 2014 Jul 16.
Vaccinia virus (VACV) L1 is an important target for viral neutralization and has been included in multicomponent DNA or protein vaccines against orthopoxviruses. To further understand the protective mechanism of the anti-L1 antibodies, we generated five murine anti-L1 monoclonal antibodies (mAbs), which clustered into 3 distinct epitope groups. While two groups of anti-L1 failed to neutralize, one group of 3 mAbs potently neutralized VACV in an isotype- and complement-independent manner. This is in contrast to neutralizing antibodies against major VACV envelope proteins such as H3, D8 or A27, which failed to completely neutralize VACV unless the antibodies are of complement-fixing isotypes and complement is present. Compared to non-neutralizing anti-L1 mAbs, the neutralization antibodies bound to the recombinant L1 protein with a significantly higher affinity and could also bind to virions. By using a variety of techniques including the isolation of neutralization escape mutants, hydrogen/deuterium exchange mass spectrometry, and X-ray crystallography, the epitope of the neutralizing antibodies was mapped to a conformational epitope with Asp35 as the key residue. This epitope is similar to the epitope of 7D11, a previously described potent VACV neutralizing antibody. The epitope was recognized mainly by CDR1 and CDR2 of the heavy chain, which are highly conserved among antibodies recognizing the epitope. These antibodies, however, had divergent light chain and heavy chain CDR3 sequences. Our study demonstrates that the conformational L1 epitope with Asp35 is a common site of vulnerability for potent neutralization by a divergent group of antibodies.Vaccinia virus, the live vaccine for smallpox, is one of the most successful vaccines in human history but presents a level of risk that has become unacceptable for the current population. Studying the immune protection mechanism of smallpox vaccine is important for understanding the basic principle of successful vaccines and the development of next generation, safer vaccines for highly pathogenic orthopoxviruses. We studied antibody targets in smallpox vaccine by developing potent neutralizing antibodies against vaccinia virus and comprehensively characterizing their epitopes. We found a site in vaccinia virus L1 protein as the target of a group of highly potent murine neutralizing antibodies. The analysis of antibody:antigen complex structure and the sequences of the antibody genes shed light on how these potent neutralizing antibodies are elicited from immunized mice.
- Glycosylated and Non-glycosylated Complement Control Protein of the Lister Strain of Vaccinia Virus. [JOURNAL ARTICLE]
- Clin Vaccine Immunol 2014 Jul 16.
The vaccinia virus complement control protein (VCP) is a secreted viral protein that binds the C3b and C4b complement components and inhibits the classical and alternative complement pathways. Previously, we reported that an attenuated smallpox vaccine, LC16m8, which was derived from the Lister strain of vaccinia virus (VV-Lister), expressed a glycosylated form of VCP, whereas published sequence data at that time indicated that the VV-Lister VCP has no motif for N-linked glycosylation. We were interested in determining whether the glycosylation of VCP impairs its biological activity, possibly contributing to the attenuation of LC16m8, and the likely origin of the glycosylated VCP. Expression analysis indicated that VV-Lister contains sub-strains expressing glycosylated VCP and those expressing non-glycosylated VCP. Other strains of smallpox vaccine, as well as laboratory strains of vaccinia virus, all expressed non-glycosylated VCP. Individual Lister virus clones expressing either the glycosylated VCP or the non-glycosylated species were isolated, and partially purified VCP from the isolates were found to be functional equivalents in binding human C3b and C4b complement proteins and inhibition of hemolysis, and in immunogenicity. Recombinant vaccinia viruses expressing FLAG-tagged glycosylated VCP (FLAG-VCPg) and non-glycosylated VCP (FLAG-VCP) were constructed based on the Western Reserve strain. Purified FLAG-VCP and FLAG-VCPg bind human C3b and C4b and blocked complement-mediated hemolysis. Our data suggest that glycosylation did not affect the biological activity of VCP, and thus may not have contributed to the attenuation of LC16m8. In addition, the LC16m8 virus likely originated from a sub-strain of VV-Lister that expresses glycosylated VCP.
- Vaccines and viral / toxin-associated neurologic infections. [JOURNAL ARTICLE]
- Handb Clin Neurol 2014.:719-744.
Vaccines have been one of the true medical milestones; implementation of vaccines has mitigated uncountable morbidity and mortality from numerous diseases, and has succeeded in eradicating one infectious disease (smallpox) from the globe. Vaccines work by stimulating the immune system to recognize pathogens or toxins and mount a response when challenged by natural infection or exposure. Vaccines have been developed against many infections and toxins resulting in neurologic disease, including bacterial meningitides, various viruses, including rabies virus, varicella-zoster virus, poliovirus, and various arthropod-borne (arbo) viruses. Despite the tremendous benefit afforded by vaccines, the immune stimulus provided by vaccination can rarely result in adverse events, including neurologic serious adverse events. This chapter will review the mechanisms by which vaccines confer immunity to various pathogens and agents, and specifically review vaccines that deal with primarily neurologic infections and illnesses. The chapter will also describe the mechanisms associated with neurologic adverse events following vaccination, and the clinical manifestations of these adverse events, as well as how to report such events to public health authorities.
- Rapid expansion of CD8+ T cells in wildtype and type I interferon receptor deficient mice correlates with protection after low-dose emergency immunization with Modified Vaccinia virus Ankara. [JOURNAL ARTICLE]
- J Virol 2014 Jul 9.
Immunization with Modified Vaccinia virus Ankara (MVA) can rapidly protect mice against lethal ectromelia virus (ECTV) infection, serving as an experimental model for severe systemic infections. Importantly, this early protective capacity of MVA vaccination completely depends on virus-specific cytotoxic CD8+ T cell responses. We used MVA vaccination in the mousepox challenge model using ECTV infection to investigate the previously unknown factors required to elicit rapid protective T cell immunity in normal C57BL/6 mice and in mice lacking the interferon alpha/beta receptor (IFNAR-/-). We found a minimal dose of 10(5) plaque-forming units of MVA vaccine fully sufficient to allow robust protection against lethal mousepox, as assessed by the absence of disease symptoms and failure to detect ECTV in organs from vaccinated animals. Moreover, MVA immunization at low dosage also protected IFNAR-/- mice, indicating efficient activation of cellular immunity even in the absence of type I interferon signaling. When monitoring for virus specific CD8+T cell responses in mice vaccinated with the minimal protective dose of MVA, we found significantly enhanced levels of antigen specific T cells in animals that were MVA vaccinated and ECTV challenged compared to mice that were only vaccinated. The initial priming of naïve CD8+ T cells by MVA immunization appears highly efficient, and even at low doses, mediates a rapid in vivo burst of pathogen-specific T cells upon challenge. Our findings define striking requirements for protective emergency immunization against severe systemic infections with orthopoxviruses.We demonstrate that single-shot low dose immunizations with vaccinia virus MVA can rapidly induce T cell mediated protective immunity against lethal orthopoxvirus infections. Our data provides new evidence for an efficient protective capacity of vaccination with replication deficient MVA. These data are of important practical relevance for public health as the effectiveness of a safety-tested next-generation smallpox vaccine based on MVA is still debated. Furthermore, producing sufficient amounts of vaccine is considered a major challenge should an outbreak occur. Moreover, prevention of other infections may require rapidly protective immunization, hence MVA could be an extremely useful vaccine for delivering heterologous T cell antigens, particularly for infectious diseases that fit a scenario of emergency vaccination.
- The genome sequence of ectromelia virus Naval and Cornell isolates from outbreaks in North America. [JOURNAL ARTICLE]
- Virology 2014 Jul 3.:218-226.
Ectromelia virus (ECTV) is the causative agent of mousepox, a disease of laboratory mouse colonies and an excellent model for human smallpox. We report the genome sequence of two isolates from outbreaks in laboratory mouse colonies in the USA in 1995 and 1999: ECTV-Naval and ECTV-Cornell, respectively. The genome of ECTV-Naval and ECTV-Cornell was sequenced by the 454-Roche technology. The ECTV-Naval genome was also sequenced by the Sanger and Illumina technologies in order to evaluate these technologies for poxvirus genome sequencing. Genomic comparisons revealed that ECTV-Naval and ECTV-Cornell correspond to the same virus isolated from independent outbreaks. Both ECTV-Naval and ECTV-Cornell are extremely virulent in susceptible BALB/c mice, similar to ECTV-Moscow. This is consistent with the ECTV-Naval genome sharing 98.2% DNA sequence identity with that of ECTV-Moscow, and indicates that the genetic differences with ECTV-Moscow do not affect the virulence of ECTV-Naval in the mousepox model of footpad infection.
- Interspecific and locational differences in metal levels in edible fish tissue from Saudi Arabia. [JOURNAL ARTICLE]
- Environ Monit Assess 2014 Jul 6.
Metal levels in fish have been extensively studied, but little data currently exists for the Middle East. We examined the levels of metals and metalloids (aluminum, arsenic, copper, manganese, selenium, zinc, and mercury) in the flesh of 13 fish species collected from three fishing sites and a local fish market in Jeddah, Saudi Arabia. We tested the following null hypotheses: (1) there are no interspecific differences in metal levels, (2) there are no differences in metal levels in fishes between market and fishing sites, (3) there are no size-related differences in metal levels, and (4) there are no differences in selenium:mercury molar ratio among different fish species. There were significant interspecific differences in concentrations for all metals. There was an order of magnitude difference in the levels of aluminum, arsenic, mercury, manganese, and selenium, indicating wide variation in potential effects on the fish themselves and on their predators. Fishes from Area II, close to a large commercial port, had the highest levels of arsenic, mercury, and selenium, followed by market fishes. Mercury was positively correlated with body size in 6 of the 13 fish species examined. Mercury was correlated positively with arsenic and selenium, but negatively with aluminum, cobalt, copper, manganese, and zinc. Selenium:mercury molar ratios varied significantly among species, with Carangoides bajad, Cephalopholis argus, Variola louti, and Ephinephelus tauvina having ratios below 10:1. These findings can be used in risk assessments, design of mercury reduction plans, development of fish advisories to protect public health, and future management decision-making.
- Safety of attenuated smallpox vaccine LC16m8 in immunodeficient mice. [JOURNAL ARTICLE]
- Clin Vaccine Immunol 2014 Jul 2.
Freeze-dried live attenuated smallpox vaccine LC16m8 prepared in cell culture has been the sole smallpox vaccine licensed in Japan since 1975 and was recently recommended as a WHO stockpile vaccine. We evaluated the safety of recently re-manufactured lots of LC16m8 using a series of immunodeficient mouse models. These models included suckling mice, severe combined immunodeficiency disease (SCID) mice, and wild-type mice treated with cyclosporine A. LC16m8 showed extremely low virulence in each of the three mouse models when compared with its parental strains, Lister and LC16mO. These results provide further evidence that LC16m8 is one of the safest replication-competent smallpox vaccines in the world, and may be considered for use in immunodeficient patients.