Protecting public health in the age of emerging infections.

Emerging and re-emerging infections cause huge concern among public health workers and international and national bodies such as the World Health Organization (WHO) and the U.S. National Institutes of Health (NIH). Indeed, scientists around the world express the view that despite the danger, research on these emerging virulent pathogens is crucial and must continue. While most of the studies underway are targeted at improving and protecting public health, some studies bear potentiallyserious risks resulting from misuse. These studies are defined as dual-use research of concern (DURC), where it is not evident that the benefits outweigh the risks. The H5N1 controversy has pushed various governments to institute new policies to govern such research. We describe the regulations that govern this emerging field of research in the United States and Israel, two countries that have taken leading stands on these issues. We suggest that the existing policies are able to mitigate many of the risks that this research encapsulates, yet more work is required--especially on the global level.

Ethical and regulatory challenges posed by synthetic biology.

Synthetic biology is a relatively new science with tremendous potential to change how we view and know the life sciences, but like many developing technologies, it has provoked ethical concerns from the scientific community and the public and confronts demands for new regulatory measures. The concerns raised involve the danger of "dual use," in which results for improving human well-being and the environment may be misappropriated for bioterror. To counteract these dangers, many governments, but the United States and Israel in particular, have introduced new laws and redoubled measures for biosafety and biosecurity. In the United States, the recent H5N1 results achieved by two groups of NIH-funded investigators highlighted the dilemma of balancing the risk of dual-use research and the freedom of science. In Israel, concern for unconventional terrorism is long-standing, and the country is constantly engaged in improving biosecurity and biodefense measures. In 2008, the Israeli parliament passed the Regulation of Research into Biological Disease Agents Law, a legislative framework for safeguarding research into biological disease agents. This article summarizes and analyzes the current state of affairs in the United States and Israel, ethical attitudes, and regulatory responses to synthetic biology.

Chimeric vaccine composed of viral peptide and mammalian heat-shock protein 60 peptide protects against West Nile virus challenge.

The protective efficacy and immunogenicity of a chimeric peptide against West Nile virus (WNV) was evaluated. This virus is the aetiological agent of West Nile fever, which has recently emerged in the western hemisphere. The rapid spread of WNV throughout North America, as well as the constantly changing epidemiology and transmission of the virus by blood transfusion and transplantation, have raised major public-health concerns. Currently, there are no effective treatments for WNV or vaccine for human use. We previously identified a novel, continuous B-cell epitope from domain III of the WNV envelope protein, termed Ep15. To test whether this epitope can protect against WNV infection, we synthesized a linear chimeric peptide composed of Ep15 and the heat-shock protein 60 peptide, p458. The p458 peptide is an effective carrier peptide for subunit vaccines against other infectious agents. We now report that mice immunized with the chimeric peptide, p458-Ep15, were resistant to lethal challenges with three different WNV strains. Moreover, their brains were free of viral genome and infectious virus. Mice immunized with Ep15 alone or with p431-Ep15, a control conjugate, were not protected. The chimeric p458-Ep15 peptide induced WNV-specific immunoglobulin G antibodies that neutralized the virus and induced the secretion of interferon-gammain vitro. Challenge of chimeric peptide-immunized mice considerably enhanced WNV-specific neutralizing antibodies. We conclude that this chimeric peptide can be used for formulation of a human vaccine against WNV.

The bioterrorism threat and dual-use biotechnological research: an Israeli perspective.

Israel has a long history of concern with chemical and biological threats, since several hostile states in the Middle East are likely to possess such weapons. The Twin-Tower terrorist attacks and Anthrax envelope scares of 2001 were a watershed for public perceptions of the threat of unconventional terror in general and of biological terror in particular. New advances in biotechnology will only increase the ability of terrorists to exploit the burgeoning availability of related information to develop ever-more destructive bioweapons. Many areas of modern biological research are unavoidably dual-use by nature. They thus have a great potential for both help and harm; and facilitating the former while preventing the latter remains a serious challenge to researchers and governments alike. This article addresses how Israel might best (1) prevent hostile elements from obtaining, from Israel's biological research system, materials, information and technologies that might facilitate their carrying out a biological attack, while (2) continuing to promote academic openness, excellence and other hallmarks of that system. This important and sensitive issue was assessed by a special national committee, and their recommendations are presented and discussed. One particularly innovative element is the restructuring and use of Israel's extensive biosafety system to also address biosecurity goals, with minimal disruption or delay.

NKp44 receptor mediates interaction of the envelope glycoproteins from the West Nile and dengue viruses with NK cells.

Dengue virus (DV) and West Nile virus (WNV) have become a global concern due to their widespread distribution and their ability to cause a variety of human diseases. Antiviral immune defenses involve NK cells. In the present study, we investigated the interaction between NK cells and these two flaviviruses. We show that the NK-activating receptor NKp44 is involved in virally mediated NK activation through direct interaction with the flavivirus envelope protein. Recombinant NKp44 directly binds to purified DV and WNV envelope proteins and specifically to domain III of WNV envelope protein; it also binds to WNV virus-like particles. These WNV-virus-like particles and WNV-domain III of WNV envelope protein directly bind NK cells expressing high levels of NKp44. Functionally, interaction of NK cells with infective and inactivated WNV results in NKp44-mediated NK degranulation. Finally, WNV infection of cells results in increased binding of rNKp44 that is specifically inhibited by anti-WNV serum. WNV-infected target cells induce IFN-gamma secretion and augmented lysis by NKp44-expressing primary NK cells that are blocked by anti-NKp44 Abs. Our findings show that triggering of NK cells by flavivirus is mediated by interaction of NKp44 with the flavivirus envelope protein.

Impaired cholesterol biosynthesis in a neuronal cell line persistently infected with measles virus.

Measles virus remains a substantial cause of morbidity and mortality, producing acute infection with a potential for development of viral persistence. To study the events underlying acute and persistent measles virus infection, we performed a global transcriptional analysis on murine neuroblastoma cells that were acutely or persistently infected with measles virus. In general, we found that acute infection induced significantly more gene expression changes than did persistent infection. A functional enrichment analysis to identify which host pathways were perturbed during each of these infections identified several pathways related to cholesterol biosynthesis, including cholesterol metabolic processes, hydroxymethylglutaryl-coenzyme A (CoA) reductase activity, and acetyl-CoA C-acetyltransferase activity. We also found that measles virus colocalized to lipid rafts in both acute and persistent infection models and that the majority of genes associated with cholesterol synthesis were downregulated in persistent infection relative to acute infection, suggesting a possible link with the defective viral budding in persistent infection. Further, we found that pharmacological inhibition of cholesterol synthesis resulted in the inhibition of viral budding during acute infection. In summary, persistent measles viral infection was associated with decreased cholesterol synthesis, a lower abundance of cholesterol and lipid rafts in the cell membrane, and inhibition of giant-cell formation and release of viral progeny.

Using high titer West Nile intravenous immunoglobulin from selected Israeli donors for treatment of West Nile virus infection.

BACKGROUND: West Nile Virus (WNV) is endemic in Israel and a significant level of antibodies is present in the population due to natural exposure. Anecdotal cases suggested that the presence of anti-WNV antibodies in intravenous immunoglobulin (IVIG) from Israeli donors (IVIG-IL) assisted the recovery of patients with severe WNV infection. METHODS: To enhance the therapeutic efficacy of IVIG-IL against WNV infection, OMRIX Biopharmaceuticals, Israel, have developed a strategy for selection of plasma units from a 10% fraction of Israeli blood donors with anti-WNV antibodies. Positive units were processed into pharmaceutical grade WNV IVIG (WNIG). Following inoculation with WNV, mice received i.p. injections of different doses (0.01-8 mg/mouse) of IVIG-IL or WNIG, according to the specific experimental protocol. RESULTS: WNIG was about 10 times more potent (per gr of IgG) than was regular IVIG-IL when tested by ELISA and neutralization assays. In a mouse lethal WNV infection model, prophylactic treatment with WNIG was at least 5-10-fold more potent as compared to treatment with IVIG-IL. Treatment with WNIG during active encephalitis, three or four days following WNV infection, had a significant protective effect. WNIG was also very effective in protecting immunosuppressed mice. Indeed, treatment of dexamethasone-immunosuppressed mice with 0.2 or 1.0 mg WNIG 4 h after virus infection, led to 100% survival. CONCLUSION: IVIG produced from selected plasma donated in WNV endemic regions can be used to produce WNV IVIG with superior activity for therapeutic and prophylactic measures.

Two-dose measles immunization as a strategy to eliminate measles in the Middle East and Israel.

The data presented above show that most countries of the Middle East (Israel and countries of the Eastern Mediterranean Region of the WHO) are progressing towards achieving WHO goals for improving immunization coverage for measles immunization to over 90%, along with the introduction of a two-dose immunization strategy. There has been corresponding progress in the reduction of incidence of measles. However, the goal of the WHO to eliminate measles has not been achieved and is unlikely to be in the near future.

T7 phage display of Ep15 peptide for the detection of WNV IgG.

West Nile virus (WNV) is one of the major emerging infectious diseases in North America. WNV belongs to the genus Flavivirus, and its rapid and extensive global spread has highlighted the necessity for accurate and specific assays for diagnosis of WNV infection. This study presents the first phage displayed peptide based ELISA for detection of WNV immunoglobulin G (IgG). The Ep15 epitope, derived from the WNV E protein DIII, was cloned into a T7 phage display system that was then used as recombinant antigen in a chemiluminescent ELISA format. The phage concentration was optimized at 5 x 10(10)PFU/ml and was used directly after polyethylene glycol concentration. The assay shows a limit of detection at a serum titer of 1:51,200 and a dynamic range from 1:100 to 1:2000. A screen of a panel of 66 human sera samples, and comparison with a commercial kit, revealed a sensitivity of 67% and a specificity of 100%. Considering the ease of antigen preparation, its stability and the optimum display properties of the T7 bacteriophage, it is apparent that this approach can be useful for the preparation of highly sensitive and specific anti-WNV immunoglobulin diagnostic kits.

Self HSP60 peptide serves as an immunogenic carrier for a CTL epitope against persistence of murine cytomegalovirus in the salivary gland.

Murine cytomegalovirus (MCMV) infection is associated with persistence of virus in the salivary glands, despite relatively rapid clearance of virus from the spleen. An effective immunization against MCMV should prevent such viral persistence. We previously reported that a peptide (p458) from the sequence of the 60 kDa heat shock protein (HSP60) molecule in a conjugate vaccine can provide T cell help for the induction of protecting antibody against bacterial capsular polysaccharides. We now report that the p458 peptide as a carrier peptide can also enhance the immunogenicity of a dominant CTL epitope of the MCMV pp89 antigen-89pep. We synthesized a linear combined peptide: chimeric p458-89pep. We immunized young BALB/c mice and challenged them with MCMV. We found that the p458-89pep chimeric peptide was more effective than the 89pep in inducing 89pep-specific IFN(gamma) secretion and specific CTL activity. Moreover, the p458-89pep chimeric peptide induced sustained IFN(gamma) secretion in the salivary gland specific to 89pep and only this immunization was associated with clearance of virus from the salivary gland. These results suggest that a peptide epitope of HSP60 may be advantageous as a T cell carrier peptide in the induction of specific T cell immunity against infectious agents.

Chemiluminescent optical fiber immunosensor for the detection of anti-West Nile virus IgG.

An ELISA-based optical fiber methodology developed for the detection of anti-West Nile virus IgG antibodies in serum was compared to standard colorimetric and chemiluminescent ELISA based on microtiter plates. Colorimetric ELISA was the least sensitive, especially at high titer dilutions. The fiber-optic immunosensor based on the same ELISA immunological rationale was the most sensitive technique.

Differential immune responses to primary measles-mumps-rubella vaccination in Israeli children.

Measles remains an important cause of morbidity and mortality worldwide, primarily due to problems associated with delivery of the live attenuated vaccine to susceptible populations. In some developed countries, there is concern about the effects of immunization on the immune system. In this study, we analyzed the responses of 12-month-old Bedouin and Jewish children living in Israel to routine measles-mumps-rubella (MMR) vaccination. Seroconversion to measles was 99% in Bedouin and 79% in Jewish children (P < 0.01), and that to mumps and rubella was 92 to 100% in both groups. Measles neutralizing antibody titers were higher in Bedouin (333 +/- 39 mIU/ml) than Jewish (122 +/- 60 mIU/ml) children (P < 0.002). Immunoglobulin G levels were higher in Bedouin than Jewish children (P = 0.007) and increased after vaccination (P = 0.0009). Leukocyte (P < 0.02) and lymphocyte (P = 0.04) counts were higher and CD4 lymphocyte percentages were lower (P < 0.001) in Bedouin than Jewish children before and after vaccination. Leukocyte counts and natural killer cell numbers did not change after vaccination, but lytic activity increased in Bedouin children (P < 0.005). Spontaneous proliferation of cultured peripheral blood mononuclear cells increased with vaccination, but there were no changes in the proliferative responses to phytohemagglutinin or tetanus toxoid. In summary, no adverse effects of MMR vaccination on immune function were detected. However, there were differences in underlying immunologic parameters and in response to the measles component of the vaccine between Bedouin and Jewish children. It is not known whether genetic differences or environmental exposure accounts for these differences.

Viruses and autoimmune diseases--adapting Koch's postulates.

Viral hypotheses for the aetiopathogenesis of autoimmune diseases are unproven. Indeed, a primary role for virus-induced autoimmune disease has yet to be fully established even in experimental systems. However new insights into virus-host interactions and improved technology justify fresh evaluation of these issues. We suggest modified Koch's postulates to test the viral hypothesis.

Prophylactic and therapeutic efficacy of human intravenous immunoglobulin in treating West Nile virus infection in mice.

West Nile virus (WNV) is a mosquito-borne disease found most commonly in Africa, West Asia, and the Middle East, where up to 40% of the human population possesses antibodies. It is an emerging disease in the United States. Humans infected with WNV develop a febrile illness that can progress to meningitis or encephalitis. In mice, WNV causes central nervous system infection, paralysis, encephalitis, and death. Currently, no specific therapy or vaccine has been approved for human use. We examined the prophylactic and therapeutic efficacy of pooled human plasma (PP) and intravenous immunoglobulin (IVIG) for treatment of WNV-infected mice. Full protection was achieved when the infected mice were treated with pooled plasma or IVIG obtained from healthy Israeli blood donors that contained WNV-specific antibodies. Similar treatments using PP or IVIG obtained from US blood donors had no protective effect. Recovery of the lethally infected mice was dependent on the dose and time of IVIG administration. These results indicate that antibodies play a major role in protection and recovery from WNV infection and that IVIG can be used as first-line therapy.

The effect of measles-mumps-rubella (MMR) immunization on the immune responses of previously immunized primary school children.

Current policies for measles control call for administration of a second dose of vaccine to immunize those who failed to respond to the initial dose and to boost the responses of those with low levels of antibody. However, there has been concern expressed publicly that reimmunization may have adverse immunologic consequences. To determine the effects of reimmunization on immune responses, primary school children (N=38, mean age=6.14+/-0.35 years) with documented previous measles-mumps-rubella vaccine (MMR) immunization during infancy 4-5 years earlier were studied before and 1 month after receiving MMR as a part of routine reimmunization in Beer-Sheva, Israel. A substantial number of children were seronegative to measles (24%), mumps (34%) and rubella (44%). On reimmunization all seroconverted to mumps and rubella and all but one (92%) seroconverted to measles. The geometric mean titer of measles virus neutralizing antibody increased from 171 to 724 and the greatest increases occurred in those with the lowest pre-immunization titers. Moderate increases in levels of total IgM, IgG and IgE were detected in those with increases in antibody to measles virus. After reimmunization leukocyte counts decreased significantly from (5.8 x 10(6))+/-2.3 to (3.4 x 10(6))+/-0.7 ml(-1) (P=0.0001). The percentages of both CD4(+) and CD8(+) T cells decreased while the CD4:CD8 ratio remained unchanged. The percentage of CD56(+) natural killer (NK) cells increased from 5.2+/-2.7 to 7.2+/-2.8 (P=0.01). Functional assays showed improved lymphoproliferation in response to stimulation with phytohemagglutinin and tetanus toxoid and stable NK lytic activity. Therefore, reimmunization was accompanied by decreased leukocyte counts, but leukocyte function was unchanged or improved.