Showing papers by "Suresh Mahalingam published in 2013"

Sphingosine kinase 1 in viral infections.

Abstract: Sphingosine kinase 1 (SphK1) is an enzyme that phosphorylates the lipid sphingosine to generate sphingosine-1-phosphate (S1P). S1P can act intracellularly as a signaling molecule and extracellularly as a receptor ligand. The SphK1/S1P axis has well-described roles in cell signaling, the cell death/survival decision, the production of a pro-inflammatory response, immunomodulation, and control of vascular integrity. Agents targeting the SphK1/S1P axis are being actively developed as therapeutics for cancer and immunological and inflammatory disorders. Control of cell death/survival and pro-inflammatory immune responses is central to the pathology of infectious disease, and we can capitalize on the knowledge provided by investigations of SphK1/S1P in cancer and immunology to assess its application to selected human infections. We have herein reviewed the growing literature relating viral infections to changes in SphK1 and S1P. SphK1 activity is reportedly increased following human cytomegalovirus and respiratory syncytial virus infections, and elevated SphK1 enhances influenza virus infection. In contrast, SphK1 activity is reduced in bovine viral diarrhea virus and dengue virus infections. Sphingosine analogs that modulate S1P receptors have proven useful in animal models in alleviating influenza virus infection but have shown no benefit in simian human immunodeficiency virus and lymphocytic choriomeningitis virus infections. We have rationalized a role for SphK1/S1P in dengue virus, chikungunya virus, and Ross River virus infections, on the basis of the biology and the pathology of these diseases. The increasing number of effective SphK1 and S1P modulating agents currently in development makes it timely to investigate these roles with the potential for developing modulators of SphK1 and S1P for novel anti-viral therapies.

Macrophage Migration Inhibitory Factor Receptor CD74 Mediates Alphavirus-Induced Arthritis and Myositis in Murine Models of Alphavirus Infection

Abstract: Objective Arthrogenic alphaviruses such as Ross River virus (RRV) and chikungunya virus (CHIKV) circulate worldwide. This virus class causes debilitating illnesses that are characterized by arthritis, arthralgia, and myalgia. In previous studies, we identified macrophage migration inhibitory factor (MIF) as a critical inflammatory factor in the pathogenesis of alphaviral diseases. The present study was undertaken to characterize the role of CD74, a cell surface receptor of MIF, in both RRV- and CHIKV-induced alphavirus arthritides. Methods Mouse models of RRV and CHIKV infection were used to investigate the immunopathogenesis of arthritic alphavirus infection. The role of CD74 was assessed using histologic analysis, real-time polymerase chain reaction, flow cytometry, and plaque assay. Results In comparison to wild-type mice, CD74−/− mice developed only mild clinical features and had low levels of tissue damage. Leukocyte infiltration, characterized predominantly by inflammatory monocytes and natural killer cells, was substantially reduced in the infected tissue of CD74−/− mice, but production of proinflammatory cytokines and chemokines was not decreased. CD74 deficiency was associated with increased monocyte apoptosis, but had no effect on monocyte migratory capacity. Consistent with these findings, alphaviral infection resulted in a dose-dependent up-regulation of CD74 expression in human peripheral blood mononuclear cells, and serum MIF levels were significantly elevated in patients with RRV or CHIKV infection. Conclusion CD74 appears to regulate immune responses to alphaviral infection through its effects on cellular recruitment and survival. These findings suggest that both MIF and CD74 play a critical role in mediating alphaviral disease, and blocking these factors with novel therapeutic agents could substantially ameliorate the pathologic manifestations.

Approaches to the treatment of disease induced by chikungunya virus

Abstract: Chikungunya virus, a re-emerging mosquito-borne alphavirus, causes fever, rash and persistent arthralgia/arthritis in humans. Severe outbreaks have occurred resulting in infections of millions of people in Southeast Asia and Africa. Currently there are no antiviral drugs or vaccines for prevention and treatment of chikungunya infections. Herein we report the current status of research on antiviral drugs and vaccines for chikungunya virus infections.

Call to action for dengue vaccine failure.

Abstract: To the Editor: Dengue is one of the most widespread infectious diseases globally; transmission now occurs in 128 countries. Although dengue virus (DENV) control strategies have targeted vector control and disease surveillance, the development of an effective vaccine is the holy grail of prevention. Dengue vaccine development has spanned many decades. A candidate vaccine (Sanofi Pasteur, Swiftwater, PA, USA) containing all 4 DENV serotypes is in advanced clinical testing. However, when given to school children in Thailand, this live-attenuated, tetravalent, dengue–yellow fever 17D chimeric virus vaccine showed major but incomplete efficacy against 3 of the 4 DENV serotypes (DENV 1 [61.2%], DENV-3 [81.3%], and DENV-4 [89.9%]) in the intention-to-treat group but no protection against DENV 2, the most pathogenic of the DENV serotypes (1). Two observations from the efficacy trial in Thailand provide insights into protective immunity that could greatly improve second-generation vaccines. The first observation was that a single dose of 4 live-attenuated chimeric DENVs given subcutaneously at a single site failed to raise type-specific protective immunity against the 4 DENV serotypes, and 2) doses 2 and 3 of the Sanofi Pasteur vaccine given to children over a 1-year period failed to improve efficacy outcomes. These results were obtained even though 91% of the children had circulating dengue or Japanese encephalitis antibodies before vaccination, neutralizing antibodies developed to all 4 DENVs, and neutralizing antibody titers increased 2–3 fold after 3 doses of vaccine in 80%–90% of vaccinated children. The inability of a mixture of 4 dengue chimeric viruses to elicit an initial primary neutralizing antibody response in nonhuman primates and susceptible humans was recognized during preclinical testing and explained by the phenomenon of interference (2). Although protective immunity was raised in susceptible rhesus monkeys inoculated with all 4 DENVs at a single site, inoculation of 4 chimeric dengue viruses at 1 or 2 sites did not result in neutralizing antibody responses to all 4 DENV (3). Studies on human primary immune responses to dengue infection have identified critical attachment sites on the virion for neutralizing antibodies (4). Serum samples from children given ≥1 doses of the dengue chimeric vaccine can now be tested for primary neutralizing antibody responses to each of the 4 DENVs. As an alternative, antibody-secreting cells may be isolated and their products identified by using methods as described for dengue-infected children in Nicaragua (5). Infections with 2 different DENVs can protect against severe disease during subsequent infections (6). It has therefore been assumed that persons with DENV neutralizing antibodies are protected against infection. In clinical testing of the Sanofi Pasteur vaccine, failure of multiple booster doses to show protection was unexpected because the children were already substantially immune from prior exposure to DENV or Japanese encephalitis vaccine. When the tetravalent dengue chimeric vaccine was given to partially dengue–immune children and adults in the Philippines, a broad neutralizing antibody response was observed after administration of only 2 vaccine doses (7). We believe that the unanticipated results of the dengue vaccine efficacy trial in Thailand call for new methods of assessing dengue immunity. Myeloid cells are major targets of dengue infection in humans. We and others have described the unique biologic responses when dengue virus–antibody complexes are presented to myeloid cells (8). There is evidence that DENV neutralization titers differ when the same antibodies are assayed in epithelial and Fc-receptor–bearing cells (8). Recent work suggests that primary monocytes and macrophages may not respond in exactly the same fashion to infection by DENV immune complexes (8). Few relevant studies exist in the literature, and most focused on DENV-2. Detailed studies on innate immune responses in human myeloid cells with a variety of dengue immune complexes should proceed forthwith. To our knowledge, only once has an in vitro test correctly predicted which children would be susceptible or have silent infections accompanying a second heterotypic dengue infection (9). This was determined by using serum samples collected before a second dengue infection and testing these serum samples at low dilutions for their ability to protect primary human monocytes from DENV-2 infection or antibody-dependent enhanced infection. During development of the Sanofi Pasteur tetravalent chimeric dengue vaccine, serum samples from vaccinated persons were routinely tested for neutralization of DENV in an epithelial cell line (10). In addition to assaying for antibodies directed at the quaternary site described by de Alwis et al. (4), we suggest that serum samples from vaccinated persons be tested for neutralization of all DENVs in primary human myeloid cells. Although human Fc-receptor cell lines may be convenient for assaying DENV antibodies, decisions regarding their use should be deferred until they are shown to model primary myeloid cells. Because antibody titers often wane after vaccination, the ability of serum samples from vaccinees to protect against infection of myeloid cells with the 4 DENVs should be studied over many years. Changes to in vitro systems for measuring immune responses after dengue vaccination may provide a better surrogate of protection by realigning antibody measurement systems to our contemporary understanding of the pathogenesis of this complex disease.

Dengue vaccine efficacy trial: does interference cause failure?

Abstract: Live-attenuated vaccines replicate at low concentrations and elicit protective immunity without causing disease. This strategy has proven to be successful when the vacc ine targets one pathogen, as is the case for vaccines against yellow fever and Japanese encephalitis viruses. Translation of this straightforward idea to target dengue has proven frustrating, because dengue is a complex fl aviviral disease that is caused by not one, but four antigenically distinct dengue viruses (DENV-1, 2, 3, and 4). Extension of the liveattenuated vaccine approach to dengue by mixing four monovalent attenuated dengue vaccine viruses into a tetravalent formulation was assessed by investigators at Mahidol University, Thailand, in a tetravalent dengue live-attenuated vaccine trial. With this approach, viral interference was reported, in which antibody responses to particular dengue serotypes were reduced or obliterated. Data from Sanofi ’s recent phase 2b trial of its tetravalent chimeric yellow fever-based dengue (CYD) vaccine in Thailand suggest that viral interference is a possible factor contributing to its low effi cacy. Viral interference in the tetravalent CYD vaccine was fi rst reported in 2009, when it was tested in monkeys as a precursor to human trials. In this study, neutralising antibody titres diff ered between CYD serotypes, with CYD-4 titres dominating. This dominance was associated with its higher replication potential and the predominance of CYD-4 viraemia, the basis for which is unknown. To confi rm the occurrence of interference in human beings unequivocally, ideally data for all four monovalent CYD vaccines individually and in tetravalent combination are needed in one trial. Although such data are unavailable, comparisons can be made between monovalent CYD-2 and tetravalent CYD vaccine trials reported by Sanofi in the past few years. Sanofi ’s CYD-2 monovalent DENV-2 vaccine showed excellent immunogenicity in a phase 1 trial. One injection of 5 log10 plaque-forming units of CYD-2 produced plaque reduction neutralisation test50 (PRNT50) geometric mean titres higher than 350 against DENV-2 strain 16 681, in both fl avivirus-naive (n=13) and fl avivirus-immune (n=14) adult volunteers. This result was durable for up to 1 year. By contrast, a CYD tetravalent formulation, containing 5 log10 plaque-forming units of each vaccine virus, tested in fl avivirus-seropositive adults (n=12) in a phase 1 trial in the dengue-endemic Philippines, produced somewhat lower PRNT50 titres to DENV-2 (246), 1 month after completion of a three-dose immunisation schedule spread across 1 year. In another phase 1 trial in Mexico City, where dengue is not endemic, a similar CYD tetravalent formulation elicited much lower DENV-2 neutralising titres (about 50), regardless of previous yellow fever vaccination. In another study of the CYD tetravalent vaccine in fl avivirus-naive adult volunteers (n=23), which resulted in low geometric mean titres against DENV-2 and detectable CYD-4 viraemia after one dose, the investigators emphasised the need for a three-dose immunisation schedule to reduce interference. In fact, in all these human studies and the monkey study, imbalance in neutralising antibody titres and dominance of DENV-4 viraemia and immunogenicity after the priming dose were recurring results consistent with the occurrence of interference in tetravalent CYD formulations. The absence of protective effi cacy against DENV-2 infection in Sanofi ’s phase 2b trial strongly suggests that interference limits CYD-2 replication and vaccine take. The high DENV-2 PRNT50 titres in this study, measured roughly 1 month after immunisation, probably represent cross-reactive antibodies elicited by the CYD-4 vaccine virus. Although these scored as DENV-2 neutralising antibodies in vitro, they may have contributed to the uptake of the DENV-2 strain (circulating in Thailand at the time of the phase 2b trial) in vivo, resulting in DENV-2 viraemia and compromising overall effi cacy. Although the fact that 2 year follow-up has not revealed any sign of 8 Heddini A, Cars O, Qiang S, Tomson G. Antibiotic resistance in China—a major future challenge. Lancet 2009; 373: 30. 9 Timothy DH, Owens CR, McGowan EJ Jr, et al. Infectious Diseases Society of America and the Society for Healthcare Epidemiology of America guidelines for developing an institutional program to enhance antimicrobial stewardship. Clin Infect Dis 2007; 44: 159–77. 10 Ministry of Health, People’s Republic of China. Notifi cation for the special campaign of antibitics rational use in hospitals. http://www.moh.gov.cn/ mohyzs/s3586/201203/54251.shtml (accessed Mar 16, 2012).

Methotrexate Treatment Causes Early Onset of Disease in a Mouse Model of Ross River Virus-Induced Inflammatory Disease through Increased Monocyte Production

Abstract: Part of the Togaviridae family, alphaviruses, including chikungunya virus (CHIKV), Sindbis virus (SINV) and Ross River virus (RRV), are able to cause significant inflammatory pathologies ranging from arthritis to encephalitis. Following symptomatic infection with arthritis-associated alphaviruses, patients often experience severe joint pain, affecting distal and small joints, which can last six months or longer. Recently, methotrexate (MTX), a disease modifying anti-rheumatic drug (DMARD), was used to treat patients experiencing chronic rheumatic symptoms following infection with CHIKV. Here, the effect of MTX on Ross River virus disease (RRVD) in mice was examined to better understand its therapeutic potential for alphaviral-induced musculoskeletal disease and to further our knowledge of the development of alphaviral pathologies. Using a mouse model, we analyzed the effect of MTX on RRVD. RRV disease pathogenesis in response to MTX treatment was determined by measuring levels of proinflammatory factors, cellular infiltrates, viral titer and histological analysis of infected tissues. RRV-infected mice receiving MTX treatment rapidly developed musculoskeletal disease, which correlated with a significant influx of inflammatory cell infiltrates into the skeletal muscle tissue. Although no difference was observed in the level of proinflammatory cytokines and chemokines, the viral load increased at early time points post infection in the serum and quadriceps of MTX treated mice, possibly contributing to disease pathogenesis. Results suggest that MTX treatment of acute RRVD in mice provides no therapeutic benefit and underline the importance of inflammatory monocytes in alphaviral induced arthritides.

Exacerbation of Alphaviral Arthritis and Myositis in a Mouse Model after Etanercept Treatment is due to Diminished Levels of Interferon a/b

Abstract: Little is known about the possible use of TNF-α inhibitors for treating viral arthritis and myositis caused by mosquitoborne arthritogenic alphaviruses such as Ross River virus (RRV), chikungunya virus (CHIKV), and Barmah Forest virus. Emerging alphaviruses such as CHIKV, Mayaro virus and O’nyong-nyong virus are now considered significant public health threats by the World Health Organisation (WHO), with reports of severe cases of debilitating arthritis and arthralgia due to these agents in numerous regions of the world [1]. Although alphavirus-induced arthritis is generally self-limiting, the disease can be severe and the virus can persist and establish chronic infection [1]. For example, Hourau et al. recently showed the persistence of CHIKV antigen in perivascular synovial macrophages in a patient 18 months after infection [2]. At present, there are no alphavirus vaccines or antiviral drugs available for humans. The current treatments available for symptoms caused by alphavirus disease consist of non-steroidal anti-inflammatory drugs such as aspirin or ibuprofen.

In-vitro Antiviral Activity of a Novel Phthalic Acid Ester Derivative Isolated from the Bangladeshi Mangrove Fern Acrostichumaureum

Abstract: Background: Over the past century dengue (DENV2), chikungunya (CHIKV) and human parainfluenza (hPiV3) viruses have profoundly impacted on human morbidity, mortality and the economy worldwide. Current therapy options to treat infections of these viruses have severe limitations leading to a continued search for novel drug candidates. Acrostichum aureum L. (Pteridaceae) is a mangrove fern, that has been used as a traditional medicine in Bangladesh and other various countries for a variety of diseases including infection. Objectives: Isolation and structural elucidation of novel antiviral secondary metabolites from the methanol extract of the aerial parts of A. aureum. Materials and methods: The novel phthalate acid ester was isolated (HPLC) and structurally elucidated using 1D and 2D NMR, MS and other spectroscopic methods. The compound was tested for antiviral activity against DENV2 andhPiV3in Vero cells using the fluorescent focus (FFA) assay and against CHIKV virus in LLC-MK2 cells using the plaque-forming unit assay (PFU). The activity of the isolated compound was further compared with its known derivative. Results: In this study, we report on the isolation of a novel phthalic acid ester, 2''-(methoxycarbonyl)-5''- methylpentyl 2'-methylhexyl phthalate from the aerial parts of the Bangladeshi mangrove fern Acrostichumaureum and its in vitro antiviral activity. The novel phthalate showed antiviral activity against dengue virus, human parainfluenza virus and chikungunya. The most potent activity was recorded against hPiV3 (EC 50 29.4 μM) and was slightly higher than the activity determined for the positive control BCX 2798 (EC50 44 μM). Cellulose acetate phthalate was also evaluated for antiviral activity against these viruses for the first time and was found to be inactive. Both compounds were found to be non-toxic against Vero and LLC-MK2 cells. Conclusion: This study shows that some selected phthalates have potent antiviral activity and should be further

Antivirals: Bindarit-The Future in Alphavirus Treatment

Abstract: Chikungunya virus (CHIKV) is an arthropod-borne virus in the Togaviridae Family, genus Alphavirus. A number of these viruses cause arthralgia and arthritis in humans, including CHIKV, Ross River virus (RRV), Semliki Forest virus, Sindbis virus, o’nyong-nyong virus, and Mayaro virus. CHIKV was first isolated in 1952 in Tanzania [1]. CHIKV infections present with fever, rash and arthralgia leading to arthritis. Its name means “that which bends up”, referring to the stooped posture of patients. There have been numerous outbreaks of CHIKV disease in Asia and Africa: for example, the 2005-06 CHIKV outbreak in La Reunion resulted in approximately 260,000 cases, including 237 deaths [2], and the 2006-07 Indian outbreak had 1.5 million reported cases [3]. Mutations in CHIKV have led to an extension of its vector competence and an increase in its human disease potential. For example, a recentlyacquired single mutation in an envelope protein now allows Aedes albopictus mosquitoes to transmit CHIKV as well as Aedes aegypti [4]. Current treatment is symptomatic, and there are specific therapies available. The recent extensive CHIKV outbreaks have led to renewed interest in CHIKV antivirals, with a number of candidates currently under study. Chloroquine phosphate was reported to be beneficial in CHIKV arthritis [5]. However, this was not confirmed in a subsequent clinical trial [6], and in an animal model of alphavirus infection choloroquine was shown to promote virus replication and enhance disease. Treatment with ribavirin improved the resolution of joint swelling in CHIKV patients [7], and synergy between interferon-α and ribavirin for inhibition of CHIKV replication has been reported. We have recently discovered that the small-molecule antiinflammatory drug bindarit is strongly therapeutic in a mouse model of CHIKV infection [8]. Bindarit was also highly effective in treating arthritis resulting from infection with the related alphavirus RRV [9]. In both these models of alphavirus infection, bindarit was able to reduce arthritic inflammation without having any detrimental effect on virus clearance. Bindarit has an innovative mechanism of action (selective inhibition of cytokine/chemokine production, particularly monocyte chemotactic proteins), a substantial and encouraging set of clinical tolerability data (more than 600 subjects, healthy volunteers and patients, treated up to a maximum dose of 2400 mg/day for as long as 6 months), and has the potential to be of benefit in a range of diseases. Being the most advanced of a group of proprietary compounds acting through a similar mechanism of action; bindarit is currently in clinical development for the treatment of type 2 diabetes nephropathy and for prevention of coronary in-stent restenosis. For rheumatic disease, oral treatment with bindarit confirmed the good tolerability profile of the drug and demonstrated statistically significant effects with a reduction of pain and pannus density. Bindarit was tested in lupus nephritis patients (an orphan drug status was granted by FDA in lupus nephritis) demonstrating a statistically significant reduction of albuminuria. This proof of concept evidence is consistent with animal data showing renal damage reduction and survival increase following bindarit treatment.