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ROCKVILLE, MD, United States

Boukhvalova M.S.,Virion Systems, Inc.
Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] | Year: 2010

Viral infection is normally detected either by viral culture or by PCR methods. Rarely is a combination of the two techniques used in the same study. Yet, when applied simultaneously, viral culture and PCR may reveal important features of viral biology, such as an abortive replication, as in the case of respiratory syncytial virus (RSV) infection. In this unit, we describe methods for detecting abortive RSV replication in a cotton rat model by using the plaque-forming unit assay and the real-time reverse-transcription PCR (qRT-PCR) assay. All steps of the process of monitoring viral replication in vivo are described, starting from the design of animal infection protocols. We continue on to the methods for extracting and processing lung samples for viral culture and RNA extraction, and finish with the actual methods of viral titration by the qRT-PCR and the plaque-forming unit assays. 2010 by John Wiley & Sons, Inc. Source


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 404.56K | Year: 2007

DESCRIPTION (provided by applicant): The development of a small animal model to study human immunodeficiency virus type-1 (HIV-1) infection would significantly facilitate studies of disease pathogenesis, as well as vaccine and anti-viral drug development and testing. However, HIV-1 replication is subjected to a number of species-specific restrictions at the level of cellular entry and/or post-entry. To date, no satisfactory small animal model for HIV-1 infection has been identified. The cotton rat has been a superb model for human infectious diseases. This animal is susceptible to an extraordinary spectrum of human pathogens, particularly viruses. Inspired by these observations, we have cloned and characterized a battery of more than 270 cotton rat genes of immunological and inflammatory importance, and reagents for their detection have been developed. Human immunodeficiency virus (HIV-1) was shown to infect cotton rats and infectious virus was transmitted from animal to animal by blood with low efficiency. In new studies developed during the Phase I of this SBIR, we have demonstrated that cotton rat cells expressing human co-receptors for HIV-1 (hCD4 with hCXCR4 or hCD4 with hCCR5) support HIV-1 infection (by kinetic experiments that measured the production of p24gag in cotton rat infected cells) and DNA integration (by cloning experiments that isolated chimeric DNA containing HIV-1 DNA sequences linked to cotton rat DNA sequences from infected cotton rat cells). Furthermore, we have shown that cotton rat cells are able to produce infective particles that can infect fresh human PBMCs. The data presented in our Progress Report indicate that the main blockage for HIV replication in cotton rat cells occurs during viral entrance and that it could be circumvented by the expression of HIV-1 co-receptors. Due to our success in demonstrating the feasibility of the model in vitro during phase I, our goal in phase II is to produce line(s) of transgenic cotton rats expressing hCD4 with hCXCR4 and hCD4 with hCCR5 molecules and test them for their infectivity to HIV-1. An HIV-1-permissive cotton rat could be widely used by the research community. The hypothesis to be tested is that cotton rats (S. hispidus) after engineered to produce HIV-1 co-receptors will be permissive to productive HIV-1 infection. We will test our hypothesis by producing lines of transgenic animals expressing hCD4 with hCCR5 and hCD4 with hCXCR4. Finaly, we will Test these transgenic lines of cotton rats in infection protocols using HIV-1. At the completion of the work proposed for phase II of this SBIR, we will have produced several lines of transgenic cotton rat expressing human co-receptors for HIV-1. Most importantly, we will have characterized these lines of animals in ex vivo and in vivo experiments An estimated 5 million people became infected with HIV worldwide in 2003, and as many as 3 million died from AIDS, according to the Joint United Nations Program on HIV/AIDS (UNAIDS). The total number of infected people worldwide is estimated at 40 million (and 600 new infections per hour). Preventive vaccine and prophylactic therapies against HIV infection and more efficient drugs for AIDS are not only urgently needed but they are also scientifically possible. However, one of the major obstacles in translational HIV research rests in the absence of inexpensive and efficient pre-clinical trial models. This is reflected in the fact that the 30 vaccine candidates that are currently being tested in clinical trials in 21 countries (only 2 have advanced to clinical phase III efficacy trials) are very similar to each other with nearly all based on only one hypothesis (protection by eliciting a cell mediated immune response), while other strong alternative hypotheses have been largely neglected (vaccines that induce neutralizing antibodies and live attenuated vaccines) due to monetary and logistic impediment to test a greater number of candidates in preclinical settings. Thus, several potential vaccines and therapeutics turn out to be buried in laboratory notebooks because the institutions where they were developed cannot afford to continue with non-human primate trials. The lack of a small animal model for HIV infection that can be used for screening a larger variety of candidates in preclinical studies is one of the most evident obstacles to speed up the process of developing HIV vaccines and AIDS therapies.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 438.79K | Year: 2006

DESCRIPTION (provided by applicant): Respiratory Syncytial virus (RSV) is the leading viral cause of death in children under 1 year and is an increasing cause of morbidity and mortality in transplant patients and the elderly. RSV causes upper and lower respiratory tract infections, occasionally leading to severe bronchiolitis and pneumonia. There is no safe and effective vaccine against RSV. Anti-RSV immunotherapy, although effective in prophylactic settings, does not provide any clinically beneficial outcome when applied therapeutically, indicating that RSV-induced pathology is mostly the result of the inflammatory response to infection rather than a direct viral effect. A combined antiviral and anti-inflammatory therapy might represent the most safe and efficient treatment against RSV infection. The expression of COX-2 and its products, prostaglandins and thromboxanes, has been correlated with the development of many inflammatory processes. Our recent studies in the cotton rat, the animal model of choice for previous studies of RSV immunoprophylaxis, strongly support our hypothesis that induction of COX-2 during RSV infection plays a pivotal role during RSV-induced inflammation and pathology and, conversely, that inhibition of COX-2 activity is a beneficial treatment for RSV-induced bronchiolitis. This application is designed to determine efficacy and safety profiles for COX-2 specific, non-steroidal anti-inflammatory drug (NSAID) treatment of RSV-induced lung pathology. Our hypothesis is that inhibition of COX-2 activity generated during RSV infection will be of therapeutic benefit during acute RSV disease, preventing the development of inflammation and pathology. The experiments will focus on primary RSV disease, with two goals in mind. The first will be to determine if COX-2 inhibition can by itself be an effective and safe treatment regimen during the acute phase of primary RSV infection. The second will be to determine whether treatment with COX-2-specific inhibitors can be complemented with antiviral therapy to improve the final outcome of RSV disease. These studies will involve correlating treatment of RSV-infected animals with pulmonary histopathology and inflammation.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 100.00K | Year: 2003

DESCRIPTION (provided by applicant): The development of a small animal model to study human immunodeficiency virus type-1 (HIV-1) infection would significantly facilitate studies on disease pathogenesis, as well as vaccine and anti-viral drug development and testing. However, HIV-1 replication is subjected to a number of species-specific restrictions at the level of cellular entry and/or post-entry. To date, no satisfactory small animal model for HIV-1 infection has been identified. The cotton rat has been a superb model for human infectious diseases. This animal is susceptible to an extraordinary spectrum of human pathogens, particularly viruses. Inspired by these observations, it has been cloned and characterized a battery of more than 20 cotton rat genes of immunological and inflammatory importance, and reagents for their detection has been developed. Human immunodeficiency virus (HIV-1) was shown to infect two species of cotton rats, Sigmodon hispidus and S. fulviventer and infectious virus was transmitted from animal to animal by blood. In new studies it was found that cotton rat cells (primary macrophages and a cotton rat osteosarcoma cell line) after transfection with a plasmid containing the backbone genome of HIV-1 support levels of HIV transcription analogous to those observed in human monocytes, indicating the absence of transcription blockage. Additionally, cotton rat cells became permissive to a HIV-1 pseudotyped infection when they transiently co-expressed human CD4 and CCR5 or CXCR4 chemokine receptors. The overall goal of this proposal is to generate a small animal model for HIV-1 infection by generating transgenic cotton rats expressing HIV-1 co-receptors. An HIV-1-permissive cotton rat could be widely used by the research community. At the completion of the exploratory experiments proposed for phase I of this SBIR, we will know the potential of the cotton rat as a transgenic model for HIV-1 studies, and whether is worth the time and expense to develop transgenic animals in a phase II study.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 100.00K | Year: 2003

DESCRIPTION (provided by applicant): Oligodeoxynucleotides containing certain unmethylated CpG sequences (CpG ODN) are powerful immunostimulants when co-administered with a variety of antigens, making them attractive adjuvants for prophylactic (e g infectious diseases) and therapeutic (e.g. cancer) vaccines. In addition to being useful when administered parenterally, they are also effective when applied topically to mucosal tissues. This makes them potentially useful in development of a respiratory syncytial virus (RSV) vaccine that would be heat-stable and would not require sterile needles and syringes. In addition to use in this country, such a vaccine would have enormous utility in developing countries, where the disease burden of RSV is the highest and where lack of refrigeration and sterile needles and syringes preclude other approaches. In preliminary studies we have shown that purified fusion glycoprotein (F) from RSV is not immunogenic when administered intranasally without an adjuvant; however, co-administration of F and CpG ODN (F/CpG) stimulated a highly effective immune response in the lungs of cotton rats. While this result was highly encouraging, the lungs of protected animals showed atypical histopathology reminiscent of that seen with an earlier, formalin-inactivated (FI-RSV) vaccine. Our published work on FI-RSV demonstrated that the histologic phenotype associated with vaccine-enhanced disease could be eliminated by the addition of monophosphoryl lipid A (MPL), and thus we believe that the atypical histopathology accompanying F/CpG immunization can also be eliminated through use of supplements. The goal of this Phase I application is to characterize the cytokine, chemokine and cellular responses to F/CpG, comparing them to primary RSV infection and to immunization with FI-RSV. Phase II activities would use that information to formulate a vaccine that would be both effective and safe.

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