Bertram Jacobs - US grants

Interferon synthesis and action is an important mechanism by which animals respond to and limit virus infection. Interferon treatment directly protects cells from virus infection and modulates the immune response to the infection. In addition, interferon inhibits the growth of some tumors in animals. Some of these responses to interferon treatment appear to be due to induction in cells of an eIF-2(alpha) protein kinase, which is activated by low but not high concentrations of dsRNA. Recently, several viruses have been characterized which code for inhibitors of the interferon-induced protein kinase. In addition it has recently been shown that histone is an inhibitor of this kinase. The goal of the proposed research is to purify the dsRNA-activated, interferon-induced protein kinase and to characterize the interaction of this kinase with its substrates and activators, and with several of its inhibitors, including EMC virus RNA and histone. The kinase will be purified, in its inactive form, from the tissue of animals which have been induced to synthesize interferon. The affinity of kinase, both in its inactive and preactivated forms, for its substrates eIF-2 and ATP will be characterized. A nitrocellulose filter binding assay will be developed and the purified kinase will be characterized in terms of its ability to bind to dsRNA and EMC/mengo virus RNA. The sequences on EMC/mengo virus RNA responsible for kinase inhibition and binding will be isolated and analyzed for the presence of secondary structure. The effects of any secondary structure found on the ability of EMC/mengo virus RNAs to interact with the kinase will be analyzed by site specific mutagenesis. The proposed research will lead to a better understanding of the activation and inhibition of this important protein kinase. As such it will increase our understanding of the molecular mechanism of interferon action. In addition, since strains of EMC virus which are relatively resistant to interferon treatment are diabetogenic in mice, and since other picornaviruses have been associated with juvenile onset diabetes in humans, this research may provide a better understanding of this important disease.

biomedical equipment resource; biomedical equipment purchase;

Interferons and dsRNA are anti-virals that have shown promise as agents in combatting infection with HIV. The work described in this research proposal will determine the mechanism by which interferon and dsRNA inhibit replication of HIV in acutely-infected cells. The proposed experiments will determine the site in replication of HIV in acutely-infected cells that is sensitive to treatment with interferon n. Experiments will be performed to determine if the interferoninduced pathways that have been implicated in inhibition of other viruses, the dsRNAdependent elF-2alpha protein kinase and the 2',5' oligo-adenylate synthetase/RNase L pathway, are activated in HIV-infected cells. Potential inhibitors of these pathways will be expressed in cell lines susceptible to infection with HIV to provide direct information concerning the importance of these pathways in inhibition of HIV replication. Since interferon and dsRNA are being tested in patients as a combination therapy with other anti-virals, the basic biological, biochemical, and molecular biological studies of the interactions between HIV and the interferon system proposed in this application may provide insights to allow better use of interferon, and agents that augment interferon's anti-viral activity in the clinic.

The interferon (IFN) system is one of the primary vertebrate defenses against virus-infection. A number of IFN-resistant virus have recently been characterized. The long-term objectives of the proposed research are to understand the mechanisms involved in resistance of viruses to IFN. The specific aims are to understand the role of one of the poxvirus inhibitors of the IFN system in replication of these viruses and in their resistance to IFN. Since poxviruses, which are being developed as vectors for human vaccines, can rescue other viruses from the effects of IFN, and since group C rotaviruses, which cause severe diarrhea in humans, encode a similar PKR inhibitor, this research may impact health issues. The role in virus replication and resistance to IFN of the vaccinia virus inhibitor of the IFN system will be evaluated by use of a mutant strain of virus, vPl080, that has been deleted for a gene involved in resistance IFN, the E3L gene. This gene has also been implicated in suppression of apoptosis in vaccinia virus infected cells, and can transform cells in culture. Activation of various components of the IFN system in vp1080- infected cells will be analyzed. Ectromelia virus (another poxvirus) deleted for the E3L homologue will be prepared and pathogenesis of this altered virus will be tested in mice. Structure function relationships for the protein encoded by E3L, p25, will be determined. p25 specifically binds double-stranded (ds)RNA, and shares homology with several other dsRNA-binding proteins, including the IFN-induced protein kinase, the human protein that binds to the HIV transactivation (tar) site, TRBP, and the rotavirus NSP3 protein. Structure/function relationships in this conserved dsRNA-binding domain will be determined by site-directed mutagenesis. Crystal structures for proteins bearing this domain will be determined. This work may provide light into how proteins recognize dsRNA. Other vaccinia virus-genes that may affect viral sensitivity to IFN will be isolated, by selecting for genes that can allow growth in the presence of IFN of vaccinia virus deleted the E3L gene. This work will provide insight into how the poxviruses have evolved resistance to IFN.

DESCRIPTION (provided by applicant): The aim of this application is to develop an improved vaccine for protection against smallpox. This vaccine will be based on attenuating mutations that we have identified in vaccinia virus. These mutations decrease neurovirulence of vaccinia virus 1,000 to >100,000-fold, but still provide excellent protection against challenge with a pathogenic poxvirus. These viruses are also reduced for pathogenesis in immune-compromised mice. Since encephalitis and disseminated disease in immune-compromised humans are amongst the most severe complications to use of vaccinia virus as a vaccine, these mutants should be safer to use than the currently available strains of vaccinia virus. We are proposing to insert these mutations into a strain of vaccinia virus that is appropriate for use in humans (NYCBH), under conditions that will allow use of these viruses in humans. Strains will be analyzed in mice for pathogenesis and for induction of a protective immune response.

[unreadable] DESCRIPTION (provided by applicant): The Aim of this proposal is to determine if replication competent but attenuated strains of vaccinia virus can be used as a vector to induce a broad mucosal immune response to inserted genes that encode HIV epitopes. A gene coding for a V3 loop multi-epitope polypeptide will be inserted into a non-essential locus of replication competent, attenuated strains of vaccinia virus. These viruses will be analyzed for expression of the HIV multi-epitope polypeptide and for replication in nasal tissue of infected animals. Animals will be immunized intra-nasally either with virus alone or as part of a prime-boost regime. Systemic, nasal and vaginal immune responses to the HIV multi-epitope polypeptide will be analyzed.

The Aim of this proposal is to understand the function of one of the major vaccinia virus (W) interferon (IFN)-resistance and neurovirulence genes, E3L. Since VV is the vaccine for smallpox, a potential biowarfare/bioterrorism agent, analysis of this virulence factor may lead to development of safer, more effective vaccines for defense against bioterrorism attacks. In addition, these safer, more effective strains of VV may be valuable for use of VV as a general vaccine vector. Since the E3L gene is highly conserved between VV and all strains of variola virus, the causative agent of smallpox, this work may lead to development of anti-smallpox drugs. The work described in this proposal will continue investigations into defining the roles that the biochemical characteristics of the E3L-encoded proteins play in evasion of the host defenses by VV. Mutants that separately affect each of the known biochemical characteristics associated with E3L-encoded proteins will be prepared and characterized. These well characterized mutants will then be used to determine the role of each of the biochemical characteristics of E3L in each of the known biological functions of E3L, including pathogenesis in the mouse model.Finally, suppressor mutations will be obtained and analyzed for specific mutations in E3L. The E3L system is unique in allowing analysis of the function of this important virulence gene from the molecular level to the level of pathogenesis in a whole animal. Thus, this work will lead to translation of basic molecular knowledge of E3L function into clinically relevant applications.

A grant has been awarded to Arizona State University under the direction of Dr. Thomas Dowling for the acquisition of a 48-capillary DNA sequencer/genetic analyzer, and an HPLC genotyping and DNA fragment purification system. This instrumentation is essential for efficient completion of the diversity of projects being conducted at ASU, including molecular, evolutionary, ecological, genomic, and bioinformatic studies. Acquisition of this equipment is necessary to handle both increasing demands (number of samples) by existing and new faculty, and will permit investigators to address questions not currently approachable with the existing systems. Research areas that will utilize the instrumentation include: (1) molecular genetics and the evolution of photosynthesis, (2) molecular systematics and evolution of a diversity of organisms (e.g., bacteria, fungi, plants, animals), (3) genetics and management of endangered species, (4) introgressive hybridization and evolutionary genomics, and (5) population biology. The equipment will become part of the DNA sequencing facility at the university. This facility provides support to several educational programs, such as the NSF REU and UMEB programs, which introduce students of diverse backgrounds to careers in science. Central to training these students is exposure to the newest techniques that allow us to obtain previously unobtainable answers. Therefore, addition of this equipment will allow us to better serve the undergraduates and graduates working in our laboratories by exposing them to state of the art technology and techniques.

Replication competent vaccinia virus (W), the current vaccine for smallpox, can cause severe complications after vaccination, especially in immune suppressed individuals. We are seeking to develop strains of VV that are replication competent, and thus will induce a strong immune response, without the complications associated with vaccination with the current vaccines. Thus, the Aim of this proposal is to prepare conditional mutants of VV that are either dependent on an FDA-approved drug for replication, or are treatable by an FDA-approved drug. For drug-sensitive viruses, individuals who experience complications could be treated with the FDA-approved drug. For drug-dependent viruses, should complications arise, the drug could be withdrawn from vaccinated individuals as a treatment. Drug-dependent viruses have the added advantage that they would not be able to spread in a viable form from vaccinated individuals to contacts. The relative immunogenicity and safety of these two strategies will be compared with that of a current vaccine (Dryvax) in immunocompetent mice (immunogenicity and safety), and in immunodeficient SCID mice (safety only). The most promising of these strains will be engineered into a virus background suitable for use in humans, prepared under GMP conditions and tested in chimpanzees and humans for safety and immunogenicity, compared to Dryvax.

Vaccination has been reported to provide prophylaxis when administered up to four days post-exposure to smallpox, although this effect is not well documented. We have developed a mouse model system to evaluate potential vaccines for prophylaxis after exposure to smallpox. In this model we show that several highly attenuated mutant strains of vaccinia virus can protect when administered intra-nasally one day postexposure to a lethal dose of wt vaccinia virus. In the research proposed in this application we will evaluate numerous strains of vaccinia virus for their ability to protect when administered post-exposure. Route of administration and dose will be optimized. For the most promising strains, mechanism of action will be determined and efficacy will be tested in mice against ectormelia, and in ground squirrels against monkeypox, in preparation for meeting the FDA Animal Efficacy Rule. This work will provide the pre-clinical data for filing an IND fora new post-exposure prophylactic drug for exposure to smallpox.

[unreadable] DESCRIPTION (provided by applicant): Replication competent vaccinia virus (W), the current vaccine for smallpox, can cause severe complications after vaccination, especially in immune suppressed individuals. We are seeking to develop attenuated strains of W that are replication competent, and thus will induce a strong immune response, without the complications associated with vaccination with the current vaccines. Thus, the Aim of this proposal is to further develop existing strains of vaccinia virus that contain mutations in a key innate immune evasion gene, the E3L IFN-resistance gene, as safe effective alternatives to the current and proposed smallpox vaccines. We have deleted the E3L gene from a tissue culture adapted variant of the NYCBH strain of W, ACAM2000. The relative immunogenicity, adjuvancy and safety of ACAM2000delE3L will be compared with that of ACAM2000 and Dryvax in mice (immunogenicity and safety). Efficacy will be tested against ectromelia, rabbitpox and monkeypox challenges in mice, rabbits and macaques, respectively, compared to Dryvax. The intent is to use this data to satisfy the FDA Animal Efficacy Rule, and pave the way for Phase l/ll Clinical Trials in healthy human volunteers. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Abstract PI: JACOBS, BERTRAM L. Project: 1R13AI091299-01 Title: XVII International Poxvirus, Asfivirus and Iridovirus Symposium Accession Number: 3255457 ================== NOTICE: THIS ABSTRACT WAS EXTRACTED FROM APPLICATION AND HAS NOT BEEN PROOFED BY AN SRA.WHEN THERE ARE PROBLEMS WITH THE APPLICATION SCANNING PROCESS, THE EXTRACTED TEXT MAY BE INCORRECT OR INCOMPLETE. ================== This R13 grant proposal is submitted to NIAID for support of the XVIII International Poxvirus, Asfivirus and Iridovirus Symposium, to be held from June 5-10, 2010 at the Poco Diablo Resort in Sedona, AZ. This is the premier meeting for the growing community of researchers working on pox and pox-related viruses and for scientists seeking to learn more about the impact of these viruses on basic and medical science. The 2010 conference continues a tradition of workshop-style meetings on pox and pox-related viruses held every two years. The 2010 conference is expected to attract approximately 225 participants. The Scientific Program will consist of 8 sessions of oral presentations by speakers drawn from submitted abstracts in the following subject areas: (1) Virus entry, (2) Viral transcription and gene expression, (3) Viral DNA biology and genome structure (including genomics and proteomics), (4) Virion assembly and egress, (5) Virus host cell-interaction and signaling, (6) Immunomodulation and pathogenesis, (7) Viral vectors and vaccines, and (8) Prophylaxis and therapy. The Scientific Program Committee will select speakers from submitted abstracts and will serve as Discussion leaders for these sessions. The Program will also include two poster sessions. To encourage career development of junior scientists, abstracts from the ranks of graduate students and post-docs will be given priority for presentation. The US government, through NIH, has mobilized efforts to develop an effective response to the public health concerns regarding smallpox and monkeypox and the need to rapidly translate the recent advances in poxvirus molecular biology into new, safe and effective strategies for treatment and prophylaxis of poxvirus infections. In addition, the recent emergence of an asfivirus in Eastern Europe and the recognition of iridoviruses as central players in fish and amphibian mortalities world-wide, make this a unique opportunity to bring together the group of virologists interested in the pox and pox-related viruses to discuss our progress in dealing with these important human and animal pathogens. Finally, with the recent partial success of a poxvirus-based HIV vaccine regimen, there is renewed interest in improving poxviruses as vaccine vectors. The timing of the XVIII International Poxvirus, Asfivirus and Iridovirus Symposium is ideally tailored to providing a venue for discussions of how new understanding of the basic biology of these important viruses can be translated into regimens that can improve human health and food security, highlighting the advances achieved by the research community in response to new initiatives and to continue to draw new talent into the field. Poxviruses cause human smallpox and human monkeypox, and the pox-related viruses (asfiviruses and iridoviruses) infect organisms important as human food sources. Poxviruses are also in the forefront for development of vaccine vectors to fight human and animal diseases. Thus, the XVIII International Poxvirus, Asfivirus and Iridovirus Symposium is ideally suited to providing a venue for discussions of how new understanding of the basic biology of these important viruses can be translated into regimens that can improve human health and food security.

DESCRIPTION (provided by applicant): Monkeypox virus and variola virus share about 95% homology, and cause similar diseases in humans. Yet one of the major determinants of orthopoxvirus virulence, the E3L gene, differs significantly between these two closely related viruses. The monkeypox virus homologue of the variola virus (and vaccinia virus) E3L gene, which is an important vaccinia virus innate immune evasion gene, is partially deleted. Since vaccinia virus, when expressing a similarly deleted E3L is attenuated in experimental animals, it is important to understand the role of this variant monkeypox virus innate immune evasion gene in replication and immune evasion of monkeypox virus. The research in this application will characterize the altered genes in monkeypox virus that are compensating for the partial deletion of the monkeypox virus E3L homologue. As such the work described in this application will provide important information on how monkeypox virus causes disease in animals and in humans. Since monkeypox virus is considered to be a re- emerging human public health concern, this research may provide the basis for development of better vaccines for protection against monkeypox virus infection in humans, and for the development of better treatments for people who do become infected with monkeypox virus.

Much of what we know about poxvirus innate immune evasion comes from work with the prototype orthopoxvirus, vaccinia virus (VACV). However, it is becoming clear that what we know for VACV is not always true for other poxviruses. The example we have been working on is monkeypox virus (MPXV). VACV has an E3L gene, which is essential for interferon-resistance, both in cells in culture and in the animal model. Despite being highly pathogenic, MPXV is missing 37 residues from the N-terminus of its E3 homologue. Thus, it is surprising that MPXV is as pathogenic as it is, despite missing this essential region of the E3 innate immune evasion protein. We have shown that while the lack of an N-terminal innate immune evasion domain in MPXV allows the virus to be sensed by the host, MPXV has evolved at least two apparently independent mechanisms to overcome the effects of being sensed in cells. The goals of this research are to understand how this unique human pathogen is sensed in infected cells and how it has evolved to counter the effects of sensing. Loss of this innate immune evasion domain makes vaccination against MPXV problematic. The final goal of this project is to develop a vaccine that can safely protect against MPXV infection.