Siddhartha Mahanty

Cutting Edge: Impairment of Dendritic Cells and Adaptive Immunity by Ebola and Lassa Viruses

Acute infection of humans with Ebola and Lassa viruses, two principal etiologic agents of hemorrhagic fevers, often results in a paradoxical pattern of immune responses: early infection, characterized by an outpouring of inflammatory mediators such as TNF-α, IL-1β, and IL-6, vs late stage infections, which are associated with poor immune responses. The mechanisms underlying these diverse outcomes are poorly understood. In particular, the role played by cells of the innate immune system, such as dendritic cells (DC), is not known. In this study, we show that Ebola and Lassa viruses infect human monocyte-derived DC and impair their function. Monocyte-derived DC exposed to either virus fail to secrete proinflammatory cytokines, do not up-regulate costimulatory molecules, and are poor stimulators of T cells. These data represent the first evidence for a mechanism by which Ebola and Lassa viruses target DC to impair adaptive immunity.

Cytokine control of parasite-specific anergy in human lymphatic filariasis. Preferential induction of a regulatory T helper type 2 lymphocyte subset.

The immunological mechanisms involved in maintenance of an asymptomatic microfilaremic state (MF) in patients with lymphatic filariasis remain undefined. MF patients have impaired filarial antigen (Ag)-specific lymphocyte proliferation and decreased frequencies (Fo) of Ag-specific T cells, and yet elevated serum IgE and antifilarial IgG4. To investigate the mechanism of Ag-specific anergy in MF patients in contrast to amicrofilaremic individuals with chronic lymphatic obstruction (CP), the Fo of Ag-specific lymphocytes from peripheral blood mononuclear cells secreting either IL-4 or IFN-gamma were assessed by filter spot enzyme-linked immunosorbent assay, and IL-10 and transforming growth factor-beta (TGF-beta) mRNA transcript levels were assessed by a semiquantitative reverse transcriptase polymerase chain reaction technique. The Fo of filaria-specific IL-4-secreting lymphocytes were equivalent in both MF (geometric mean [GM] = 1:11,700) and CP (GM = 1:29,300 P = 0.08), whereas the Fo of IFN-gamma-secreting lymphocytes were lower in MF (GM = 1:39,300) than in CP (GM = 1:4,200, P < 0.01). When the ratio of IL-4/IFN-gamma (T helper type 2 [Th2]/Th1)-secreting cells was examined, MF subjects showed a predominant Th2 response (8:1) compared with a Th1 response in CP individuals (1:4). mRNA transcript levels of IL-10 were also significantly elevated in MF compared with CP individuals (P < 0.01). Further, IL-10 and TGF-beta were shown to have a role in modulating the Ag-specific anergy among MF subjects, in that neutralizing anti-IL-10 or anti-TGF-beta significantly enhanced lymphocyte proliferation response (by 220-1,300%) to filarial Ags in MF individuals. These findings demonstrate that MF subjects respond to parasite antigen by producing a set of suppressive cytokines that may facilitate persistence of the parasite within humans while producing little clinical disease.

Analysis of Human Peripheral Blood Samples from Fatal and Nonfatal Cases of Ebola (Sudan) Hemorrhagic Fever: Cellular Responses, Virus Load, and Nitric Oxide Levels

ABSTRACT Peripheral blood samples obtained from patients during an outbreak of Ebola virus (Sudan species) disease in Uganda in 2000 were used to phenotype peripheral blood mononuclear cells (PBMC), quantitate gene expression, measure antigenemia, and determine nitric oxide levels. It was determined that as the severity of disease increased in infected patients, there was a corresponding increase in antigenemia and leukopenia. Blood smears revealed thrombocytopenia, a left shift in neutrophils (in some cases degenerating), and atypical lymphocytes. Infected patients who died had reduced numbers of T cells, CD8+ T cells, and activated (HLA-DR+) CD8+ T cells, while the opposite was noted for patients who survived the disease. Expression levels of cytokines, Fas antigen, and Fas ligand (TaqMan quantitation) in PBMC from infected patients were not significantly different from those in uninfected patients (treated in the same isolation wards), nor was there a significant increase in expression compared to healthy volunteers (United States). This unresponsive state of PBMC from infected patients despite high levels of circulating antigen and virus replication suggests that some form of immunosuppression had developed. Ebola virus RNA levels (virus load) in PBMC specimens were found to be much higher in infected patients who died than patients who survived the disease. Similarly, blood levels of nitric oxide were much higher in fatal cases (increasing with disease severity), and extremely elevated levels (≥150 μM) would have negatively affected vascular tone and contributed to virus-induced shock.

Phase 1 Clinical Trial of Apical Membrane Antigen 1: an Asexual Blood-Stage Vaccine for Plasmodium falciparum Malaria

ABSTRACT Apical membrane antigen 1 (AMA1), a polymorphic merozoite surface protein, is a leading blood-stage malaria vaccine candidate. A phase 1 trial was conducted with 30 malaria-naïve volunteers to assess the safety and immunogenicity of the AMA1-C1 malaria vaccine. AMA1-C1 contains an equal mixture of recombinant proteins based on sequences from the FVO and 3D7 clones of Plasmodium falciparum. The proteins were expressed in Pichia pastoris and adsorbed on Alhydrogel. Ten volunteers in each of three dose groups (5 μg, 20 μg, and 80 μg) were vaccinated in an open-label study at 0, 28, and 180 days. The vaccine was well tolerated, with pain at the injection site being the most commonly observed reaction. Anti-AMA1 immunoglobulin G (IgG) was detected by enzyme-linked immunosorbent assay (ELISA) in 15/28 (54%) volunteers after the second immunization and in 23/25 (92%) after the third immunization, with equal reactivity to both AMA1-FVO and AMA1-3D7 vaccine components. A significant dose-response relationship between antigen dose and antibody response by ELISA was observed, and the antibodies were predominantly of the IgG1 isotype. Confocal microscopic evaluation of sera from vaccinated volunteers demonstrated reactivity with P. falciparum schizonts in a pattern similar to native parasite AMA1. Antigen-specific in vitro inhibition of both FVO and 3D7 parasites was achieved with IgG purified from sera of vaccinees, demonstrating biological activity of the antibodies. To our knowledge, this is the first AMA1 vaccine candidate to elicit functional immune responses in malaria-naïve humans, and our results support the further development of this vaccine.

Pathogenesis of filoviral haemorrhagic fevers

The filoviruses, marburgvirus and ebolavirus, cause epidemics of haemorrhagic fever with high case-fatality rates. The severe illness results from a complex of pathogenetic mechanisms that enable the virus to suppress innate and adaptive immune responses, infect and kill a broad variety of cell types, and elicit strong inflammatory responses and disseminated intravascular coagulation, producing a syndrome resembling septic shock. Most experimental data have been obtained on Zaire ebolavirus, which causes uniformly lethal disease in experimentally infected non-human primates but produces a broader range of outcomes in naturally infected human beings. 10-30% of patients can survive the illness by mobilising adaptive immune responses, and there is limited evidence that mild or symptomless infections also occur. The other filoviruses that have caused human disease, Sudan ebolavirus, Ivory Coast ebolavirus, and marburgvirus, produce a similar illness but with somewhat lower case-fatality rates. Variations in outcome during an epidemic might be due partly to genetically determined differences in innate immune responses to the viruses. Recent studies in non-human primates have shown that blocking of certain host responses, such as the coagulation cascade, can result in reduced viral replication and improved host survival.

Passive Transfer of Antibodies Protects Immunocompetent and Immunodeficient Mice against Lethal Ebola Virus Infection without Complete Inhibition of Viral Replication

ABSTRACT Ebola hemorrhagic fever is a severe, usually fatal illness caused by Ebola virus, a member of the filovirus family. The use of nonhomologous immune serum in animal studies and blood from survivors in two anecdotal reports of Ebola hemorrhagic fever in humans has shown promise, but the efficacy of these treatments has not been demonstrated definitively. We have evaluated the protective efficacy of polyclonal immune serum in a mouse model of Ebola virus infection. Our results demonstrate that mice infected subcutaneously with live Ebola virus survive infection and generate high levels of anti-Ebola virus immunoglobulin G (IgG). Passive transfer of immune serum from these mice before challenge protected upto 100% of naive mice against lethal Ebola virus infection. Protection correlated with the level of anti-Ebola virus IgG titers, and passive treatment with high-titer antiserum was associated with a delay in the peak of viral replication. Transfer of immune serum to SCID mice resulted in 100% survival after lethal challenge with Ebola virus, indicating that antibodies alone can protect from lethal disease. Thus antibodies suppress or delay viral growth, provide protection against lethal Ebola virus infection, and may not require participation of other immune components for protection.

Cytokine Production in Acute versus Chronic Human Schistosomiasis Mansoni: The Cross-Regulatory Role of Interferon-γ and Interleukin-10 in the Responses of Peripheral Blood Mononuclear Cells and Splenocytes to Parasite Antigens

The contribution of interleukin (IL)-10 and interferon (IFN)-gamma to the regulation of type 1 and type 2 cytokine responses was investigated in Brazilians with different clinical forms of schistosomiasis mansoni. Cells from members of a family with acute intestinal schistosomiasis responded to schistosomal soluble egg antigen (SEA) or soluble adult worm antigen preparation (SWAP) with greater amounts of IFN-gamma than did cells from several patients with chronic intestinal schistosomiasis; IL-10 levels were similar. Neutralization of IL-10 had no effect on the SEA-specific IFN-gamma response in patients with acute infection, whereas SWAP-induced IFN-gamma was increased in both groups. Anti-IL-10 also up-regulated SEA-specific IFN-gamma protein and mRNA responses in most splenocyte cultures from hepatosplenic schistosomiasis patients but had no effect on antigen-specific IL-4 or IL-5 production. Neutralization of IFN-gamma resulted in a comparable increase in SWAP-specific IL-10 and IL-5, while IL-4 was not affected. These studies demonstrate that early disease in schistosomiasis is associated with a significant IFN-gamma response and that IL-10 contributes to the suppression of that response during both early and chronic infection.

Immunoregulation in human lymphatic filariasis: the role of interleukin 10.

In humans with lymphatic filariasis microfilaremia is associated with a parasite antigen‐specific hyporesponsive‐ness when assessed by cell proliferation and secretion of interleukin‐2 and interferon‐γ. Hyporesponsiveness in these individuals is not only parasite antigen‐specific but appears to be limited to Th1‐type responses. Th2 mediated responses such as IL‐5 secretion and IgE antibody production to parasite antigens are generally strong and usually no different than those seen in immunologically more reactive amicrofilaremic individuals with chronic lymphatic pathology. The mechanisms by which Th1 responses are inhibited have not yet been elucidated, but some studies suggest that down‐regulatory cytokines such as IL‐10 may be involved in this process. Mononuclear cells from microfilaremic individuals have been found to secrete greater quantities of IL‐10 spontaneously and in response to parasite antigens. In this review, mechanisms by which IL‐10 may be induced by the parasite and the mode by which IL‐10 may regulate parasite antigen‐specific Th1 responses in these individuals are discussed.

Low Levels of Interleukin-8 and Interferon-Inducible Protein–10 in Serum Are Associated with Fatal Infections in Acute Lassa Fever

To investigate the role of inflammatory mediators in the pathogenesis of Lassa fever, the levels of a number of pro- and anti-inflammatory cytokines and chemokines in serum samples collected from hospitalized patients with fatal and nonfatal acute Lassa fever were compared with those from 2 control groups: patients with other febrile illnesses and uninfected individuals. Serum interleukin (IL)-8 and interferon (IFN)-inducible protein (IP)-10 levels were significantly higher in patients with acute nonfatal Lassa fever than in control subjects. In striking contrast, levels of these chemokines were low or undetectable in patients with fatal Lassa fever. IFN-gamma, IL-12, IL-6, and RANTES levels were elevated in all the febrile study groups. Tumor necrosis factor-alpha levels were not elevated in patients with fatal or nonfatal Lassa fever. These data indicate that acute nonfatal Lassa fever is associated with high levels of circulating IL-8 and IP-10 and that low levels or absence of these mediators correlates with a poor outcome.

Ebola Hemorrhagic Fever and Septic Shock

Ebola virus is the cause of sporadic outbreaks of lethal Ebola hemorrhagic fever (EHF) in central Africa. Despite the difficulties of studying this virus much has been learned over the past decade about the pathogenesis of Ebola virus infection in humans and nonhuman primates. Two articles in this issue of the Journal of Infectious Diseases report further progress. The article by Bosio et al. confirms findings that the virus is able to infect dendritic cells (DCs) impairing their innate antiviral activity and limiting their ability to initiate adaptive immune responses. The article by Geisbert et al. identifies a molecular trigger for disseminated intravascular coagulation (DIC) through the expression of tissue factor (TF) on the surface of virus-infected monocytes and macrophages. Together these findings shed light on early events that both permit rapid viral dissemination and cause some of the major features of EHF. (excerpt)