Mutating the CX3C Motif in the G Protein Should Make a Live Respiratory Syncytial Virus Vaccine Safer and More Effective.

Respiratory syncytial virus (RSV) belongs to the family Paramyxoviridae and is the single most important cause of serious lower respiratory tract infections in young children, yet no highly effective treatment or vaccine is available. Through a CX3C chemokine motif (182CWAIC186) in the G protein, RSV binds to the corresponding chemokine receptor, CX3CR1. Since RSV binding to CX3CR1 contributes to disease pathogenesis, we investigated whether a mutation in the CX3C motif by insertion of an alanine, A186, within the CX3C motif, mutating it to CX4C (182CWAIAC187), which is known to block binding to CX3CR1, might decrease disease. We studied the effect of the CX4C mutation in two strains of RSV (A2 and r19F) in a mouse challenge model. We included RSV r19F because it induces mucus production and airway resistance, two manifestations of RSV infection in humans, in mice. Compared to wild-type (wt) virus, mice infected with CX4C had a 0.7 to 1.2 log10-fold lower virus titer in the lung at 5 days postinfection (p.i.) and had markedly reduced weight loss, pulmonary inflammatory cell infiltration, mucus production, and airway resistance after challenge. This decrease in disease was not dependent on decrease in virus replication but did correspond to a decrease in pulmonary Th2 and inflammatory cytokines. Mice infected with CX4C viruses also had higher antibody titers and a Th1-biased T cell memory response at 75 days p.i. These results suggest that the CX4C mutation in the G protein could improve the safety and efficacy of a live attenuated RSV vaccine.IMPORTANCE RSV binds to the corresponding chemokine receptor, CX3CR1, through a CX3C chemokine motif (182CWAIC186) in the G protein. RSV binding to CX3CR1 contributes to disease pathogenesis; therefore, we investigated whether a mutation in the CX3C motif by insertion of an alanine, A186, within the CX3C motif, mutating it to CX4C (182CWAIAC187), known to block binding to CX3CR1, might decrease disease. The effect of this mutation and treatment with the F(ab')2 form of the anti-RSV G 131-2G monoclonal antibody (MAb) show that mutating the CX3C motif to CX4C blocks much of the disease and immune modulation associated with the G protein and should improve the safety and efficacy of a live attenuated RSV vaccine.

CD8-deficient SJL mice display enhanced susceptibility to Theiler's virus infection and increased demyelinating pathology.

Theiler's murine encephalomyelitis virus (TMEV) infection of the central nervous system (CNS) induces a chronic, progressive demyelinating disease in susceptible mouse strains characterized by inflammatory mononuclear infiltrates and spastic hind limb paralysis. Our lab has previously demonstrated a critical role for TMEV- and myelin-specific CD4(+) T cells in initiating and perpetuating this pathology. It has however, also been shown that the MHC class I loci are associated with susceptibility/resistance to TMEV infection and persistence. For this reason, we investigated the contribution of CD8(+) T cells to the TMEV-induced demyelinating pathology in the highly susceptible SJL/J mouse strain. Here we show that beta2M-deficient SJL mice have similar disease incidence rates to wild-type controls, however beta2M-deficient mice demonstrated earlier onset of clinical disease, elevated in vitro responses to TMEV and myelin proteolipid (PLP) epitopes, and significantly higher levels of CNS demyelination and macrophage infiltration at 50 days post-infection. beta2M-deficient mice also displayed a significant elevation in persisting viral titers, as well as an increase in macrophage-derived pro-inflammatory cytokine mRNA expression in the spinal cord at this same time point. Taken together, these results indicate that CD8(+) T cells are not required for clinical or histologic disease initiation or progression in TMEV-infected SJL mice. Rather, these data stress the critical role of CD4(+) T cells in this capacity and further emphasize the potential for CD8(+) T cells to contribute to protection from TMEV-induced demyelination.

Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus.

The mammalian Toll-like receptor 4, TLR4, is an important component in the innate immune response to gram-negative bacterial infection. The role of TLR4 in antiviral immunity has been largely unexplored. In this study, the in vivo immune responses to respiratory syncytial virus (RSV) and influenza virus infection were examined in TLR4-deficient (C57BL/10ScNCr) and TLR4-expressing (C57BL/10Sn) mice. TLR4-deficient mice challenged with RSV, but not influenza virus, exhibited impaired natural killer (NK) cell and CD14(+) cell pulmonary trafficking, deficient NK cell function, impaired interleukin-12 expression, and impaired virus clearance compared to mice expressing TLR4. These findings suggest that Toll signaling pathways have an important role in innate immunity to RSV.

CX3C chemokine mimicry by respiratory syncytial virus G glycoprotein.

Chemokines are chemoattractant proteins that are divided into subfamilies based upon cysteine signature motifs termed C, CC, CXC and CX3C. Chemokines have roles in immunity and inflammation that affect cell trafficking and activation of T cells as well as cells of the innate immune system. We report here CX3C chemokine mimicry for the G glycoprotein of respiratory syncytial virus (RSV) and show binding to CX3CR1--the specific receptor for the CX3C chemokine fractalkine--and induction of leukocyte chemotaxis. We also show that CX3CR1 facilitates RSV infection of cells. Thus, G glycoprotein interaction with CX3CR1 probably plays a key role in the biology of RSV infection.

CD8(+) T cells from Theiler's virus-resistant BALB/cByJ mice downregulate pathogenic virus-specific CD4(+) T cells.

Theiler's murine encephalomyelitis virus (TMEV) is a picornavirus which induces an immune-mediated demyelinating disease in susceptible strains of mice and serves as a relevant animal model for multiple sclerosis. Treatment with low dose irradiation prior to infection with the BeAn strain of TMEV renders the genetically resistant BALB/cByJ (C/cByJ) mice susceptible to disease. Previous studies have shown that disease resistance in the C/cByJ is mediated by a 'regulatory' CD8(+) T cell population, which does not appear to function via a cytolytic mechanism. We show here that TMEV-specific CD4(+) T cell blasts transferred into susceptible, irradiated C/cByJ accelerate clinical disease and enhance TMEV-specific DTH and proliferation in these animals. Significantly, CD8(+) cells from infected, resistant C/cByJ mice specifically downregulate the in vivo disease potentiation and diminish virus specific DTH, and proliferative and pro-inflammatory cytokine responses (IFNgamma and IL-2) in recipients of TMEV-specific CD4(+) T cell blasts. These results indicate that TMEV infection of resistant C/cByJ mice induces a radiosensitive population of regulatory CD8(+) T cells which actively downregulate inherent Th1 responses which have disease initiating potential.

Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus.

The innate immune system contributes to the earliest phase of the host defense against foreign organisms and has both soluble and cellular pattern recognition receptors for microbial products. Two important members of this receptor group, CD14 and the Toll-like receptor (TLR) pattern recognition receptors, are essential for the innate immune response to components of Gram-negative and Gram-positive bacteria, mycobacteria, spirochetes and yeast. We now find that these receptors function in an antiviral response as well. The innate immune response to the fusion protein of an important respiratory pathogen of humans, respiratory syncytial virus (RSV), was mediated by TLR4 and CD14. RSV persisted longer in the lungs of infected TLR4-deficient mice compared to normal mice. Thus, a common receptor activation pathway can initiate innate immune responses to both bacterial and viral pathogens.

The role of protective CD8+ T cells in resistance of BALB/c mice to Theiler's murine encephalomyelitis virus-induced demyelinating disease: regulatory vs. lytic.

Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease (TMEV-IDD) is an excellent model for human multiple sclerosis. Within the BALB/c strain, BALB/cAnNCr mice are susceptible while BALB/cByJ mice are resistant. BALB/cByJ mice become susceptible when irradiated. Adoptive transfer of CD8+ splenic T cells from resistant BALB/cByJ donors protect irradiated BALB/cByJ, as well as BALB/cAnNCr recipients, from development of TMEV-IDD. Anti-TMEV CTL activities in BALB/cAnNCr, BALB/cByJ and irradiated BALB/cByJ mice are comparable. A population of splenic CD4+ T cells in BALB/cByJ donors has also been identified which can protect both susceptible BALB/cAnNCr and irradiated BALB/cByJ recipients from TMEV-IDD via adoptive transfer.

Drug information resources in the Veterans Affairs healthcare system.

Results of a survey characterizing drug information services and resources from a defined practice area are presented. At the end of 1992, a questionnaire was mailed to 167 Veterans Affairs Medical Centers and Outpatient Clinics. One hundred fifty-one of the surveys (> 90%) were completed and analyzed. The knowledge obtained from this survey may provide a basis for future development of an essential clinical service within the Veterans Affairs health care system.

Drug information service for drug product procurement in the Veterans Affairs health-care system: preliminary experience.

The preliminary experience of the drug information service of the Department of Veterans Affairs (VA) central office is described. The drug information service assists the drug and pharmaceutical product management section of the pharmacy service for the VA central office. The purpose of the drug information service is to promote efficacious drug therapy while meeting cost containment goals for pharmaceutical products. The pharmacist coordinator of this service has experience in both patient care and drug information service. The drug information service is involved in the following activities: (1) making recommendations for contract bidding on therapeutically equivalent products, (2) identifying prescription duplication within the system, (3) reporting product defects, (4) planning drug procurement in unique situations, such as during war, (5) developing gender-specific therapy, (6) evaluating the appropriateness of brand-name-only purchasing of certain products, (7) evaluating new drug products, (8) compiling national drug-use data, and (9) projecting drug price increases. The VA drug information service has diverse responsibilities meant to optimize drug therapy and reduce pharmacy costs in the VA health-care system.