Sergio Gómez-Medina

Cytokine kinetics of Zika virus-infected patients from acute to reconvalescent phase

AbstractnZika virus is an emerging mosquito-borne flavivirus currently causing large epidemics in the Pacific Ocean region and Brazil. Clinically, Zika fever resembles dengue fever, but is less severe. Whereas the clinical syndrome and laboratory diagnostic procedures have been described, little attention was paid to the immunology of the disease and its possible use for clinical follow-up of patients. Here, we investigate the role of cytokines in the pathogenesis of Zika fever in travelers returning from Asia, the Pacific, and Brazil. Polyfunctional T cell activation (Th1, Th2, Th9, and Th17 response) was seen during the acute phase characterized by respective cytokine level increases, followed by a decrease in the reconvalescent phase.

Mucosal Polyinosinic-Polycytidylic Acid Improves Protection Elicited by Replicating Influenza Vaccines via Enhanced Dendritic Cell Function and T Cell Immunity

Live-attenuated influenza vaccines (LAIVs) have the potential to generate CD8 T cell immunity that may limit the virulence of an antigenically shifted influenza strain in a population lacking protective Abs. However, current LAIVs exert limited T cell immunity restricted to the vaccine strains. One approach to improve LAIV-induced T cell responses is the use of specific adjuvants to enhance T cell priming by respiratory dendritic cells, but this hypothesis has not been addressed. In this study, we assessed the effect of the TLR3 ligand polyinosinic-polycytidylic acid (poly IC) on CD8 T cell immunity and protection elicited by LAIVs. Mucosal treatment with poly IC shortly after vaccination enhanced respiratory dendritic cell function, CD8 T cell formation, and production of neutralizing Abs. This adjuvant effect of poly IC was dependent on amplification of TLR3 signaling by nonhematopoietic radioresistant cells and enhanced mouse protection to homosubtypic, as well as heterosubtypic, virus challenge. Our findings indicate that mucosal TLR3 ligation may be used to improve CD8 T cell responses to replicating vaccines, which has implications for protection in the absence of pre-existing Ab immunity.

Ebola Virus Disease in Mice with Transplanted Human Hematopoietic Stem Cells

ABSTRACT The development of treatments for Ebola virus disease (EVD) has been hampered by the lack of small-animal models that mimick human disease. Here we show that mice with transplanted human hematopoetic stem cells reproduce features typical of EVD. Infection with Ebola virus was associated with viremia, cell damage, liver steatosis, signs of hemorrhage, and high lethality. Our study provides a small-animal model with human components for the development of EVD therapies.

Novel Cross-Reactive Monoclonal Antibodies against Ebolavirus Glycoproteins Show Protection in a Murine Challenge Model

ABSTRACT Out of an estimated 31,100 cases since their discovery in 1976, ebolaviruses have caused approximately 13,000 deaths. The vast majority (∼11,000) of these occurred during the 2013-2016 West African epidemic. Three out of five species in the genus are known to cause Ebola Virus Disease in humans. Several monoclonal antibodies against the ebolavirus glycoprotein are currently in development as therapeutics. However, there is still a paucity of monoclonal antibodies that can cross-react between the glycoproteins of different ebolavirus species, and the mechanism of these monoclonal antibody therapeutics is still not understood in detail. Here, we generated a panel of eight murine monoclonal antibodies (MAbs) utilizing a prime-boost vaccination regimen with a Zaire ebolavirus glycoprotein expression plasmid followed by infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. We tested the binding breadth of the resulting monoclonal antibodies using a set of recombinant surface glycoproteins from Reston, Taï Forest, Bundibugyo, Zaire, Sudan, and Marburg viruses and found two antibodies that showed pan-ebolavirus binding. An in vivo Stat2−/− mouse model was utilized to test the ability of these MAbs to protect from infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. Several of our antibodies, including the broadly binding ones, protected mice from mortality despite lacking neutralization capability in vitro, suggesting their protection may be mediated by Fc-FcR interactions. Indeed, three antibodies displayed cellular phagocytosis and/or antibody-dependent cell-mediated cytotoxicity in vitro. Our antibodies, specifically the two identified cross-reactive monoclonal antibodies (KL-2E5 and KL-2H7), might add to the understanding of anti-ebolavirus humoral immunity. IMPORTANCE This study describes the generation of a panel of novel anti-ebolavirus glycoprotein monoclonal antibodies, including two antibodies with broad cross-reactivity to all known ebolavirus species. The antibodies were raised using a heterologous DNA-viral vector prime-boost regimen, resulting in a high proportion of cross-reactive antibodies (25%). Similar vaccination regimens have been used successfully to induce broad protection against influenza viruses in humans, and our limited data indicate that this might be a useful strategy for filovirus vaccines as well. Several of our antibodies showed protective efficacy when tested in a novel murine challenge model and may be developed into future therapeutics.

Ebola virus infection kinetics in chimeric mice reveal a key role of T cells as barriers for virus dissemination

Ebola virus (EBOV) causes severe systemic disease in humans and non-human primates characterized by high levels of viremia and virus titers in peripheral organs. The natural portals of virus entry are the mucosal surfaces and the skin where macrophages and dendritic cells (DCs) are primary EBOV targets. Due to the migratory properties of DCs, EBOV infection of these cells has been proposed as a necessary step for virus dissemination via draining lymph nodes and blood. Here we utilize chimeric mice with competent hematopoietic-driven immunity, to show that EBOV primarily infects CD11b+ DCs in non-lymphoid and lymphoid tissues, but spares the main cross-presenting CD103+ DC subset. Furthermore, depletion of CD8 and CD4 T cells resulted in loss of early control of virus replication, viremia and fatal Ebola virus disease (EVD). Thus, our findings point out at T cell function as a key determinant of EVD progress and outcome.

Distinct Immunogenicity and Efficacy of Poxvirus-based Vaccine Candidates against Ebola Virus expressing GP and VP40 Proteins

ABSTRACT Zaire and Sudan ebolavirus species cause a severe disease in humans and nonhuman primates (NHPs) characterized by a high mortality rate. There are no licensed therapies or vaccines against Ebola virus disease (EVD), and the recent 2013 to 2016 outbreak in West Africa highlighted the need for EVD-specific medical countermeasures. Here, we generated and characterized head-to-head the immunogenicity and efficacy of five vaccine candidates against Zaire ebolavirus (EBOV) and Sudan ebolavirus (SUDV) based on the highly attenuated poxvirus vector modified vaccinia virus Ankara (MVA) expressing either the virus glycoprotein (GP) or GP together with the virus protein 40 (VP40) forming virus-like particles (VLPs). In a human monocytic cell line, the different MVA vectors (termed MVA-EBOVs and MVA-SUDVs) triggered robust innate immune responses, with production of beta interferon (IFN-β), proinflammatory cytokines, and chemokines. Additionally, several innate immune cells, such as dendritic cells, neutrophils, and natural killer cells, were differentially recruited in the peritoneal cavity of mice inoculated with MVA-EBOVs. After immunization of mice with a homologous prime/boost protocol (MVA/MVA), total IgG antibodies against GP or VP40 from Zaire and Sudan ebolavirus were differentially induced by these vectors, which were mainly of the IgG1 and IgG3 isotypes. Remarkably, an MVA-EBOV construct coexpressing GP and VP40 protected chimeric mice challenged with EBOV to a greater extent than a vector expressing GP alone. These results support the consideration of MVA-EBOVs and MVA-SUDVs expressing GP and VP40 and producing VLPs as best-in-class potential vaccine candidates against EBOV and SUDV. IMPORTANCE EBOV and SUDV cause a severe hemorrhagic fever affecting humans and NHPs. Since their discovery in 1976, they have caused several sporadic epidemics, with the recent outbreak in West Africa from 2013 to 2016 being the largest and most severe, with more than 11,000 deaths being reported. Although some vaccines are in advanced clinical phases, less expensive, safer, and more effective licensed vaccines are desirable. We generated and characterized head-to-head the immunogenicity and efficacy of five novel vaccines against EBOV and SUDV based on the poxvirus MVA expressing GP or GP and VP40. The expression of GP and VP40 leads to the formation of VLPs. These MVA-EBOV and MVA-SUDV recombinants triggered robust innate and humoral immune responses in mice. Furthermore, MVA-EBOV recombinants expressing GP and VP40 induced high protection against EBOV in a mouse challenge model. Thus, MVA expressing GP and VP40 and producing VLPs is a promising vaccine candidate against EBOV and SUDV.

Humanized Mice Reproduce Acute and Persistent Human Adenovirus Infection

Severe human adenovirus (HAdV) infections are an increasing threat for immunosuppressed individuals, particularly those who have received stem cell transplants. It has been previously hypothesized that severe infections might be due to reactivation of a persistent infection, but this hypothesis has been difficult to test owing to the lack of a permissive in vivo model of HAdV infection. Here we established a humanized mouse model that reproduces features of acute and persistent HAdV infection. In this model, acute infection correlated with high mortality, weight loss, liver pathology, and expression of viral proteins in several organs. In contrast, persistent infection was asymptomatic and led to establishment of HAdV-specific adaptive immunity and expression of early viral genes exclusively in the bone marrow. These findings validate the use of humanized mice to study acute and persistent HAdV infection and strongly suggest the presence of cellular reservoirs in the bone marrow.

Monocyte-derived dendritic cells enhance protection against secondary influenza challenge by controlling the switch in CD8(+) T-cell immunodominance.

Influenza virus infection triggers an increase in the number of monocyte‐derived dendritic cells (moDCs) in the respiratory tract, but the role of these cells during antiviral immunity is still unclear. Here we show that during influenza infection, moDCs dominate the late activation of CD8+ T cells and trigger the switch in immunodominance of the CD8+ T‐cell response from acidic polymerase specificity to nucleoprotein specificity. Abrogation of monocyte recruitment or depletion of moDCs strongly compromised host resistance to secondary influenza challenge. These findings underscore a novel function of moDCs in the antiviral response to influenza virus, and have important implications for vaccine design.

Sustained Elevated Cytokine Levels during Recovery Phase of Mayaro Virus Infection.

To the Editor: Mayaro virus (MAYV), a mosquitoborne alphavirus endemic to South America, causes a self-limiting febrile arthralgia syndrome closely resembling Chikungunya fever (1). MAYV has been detected increasingly as imported infections in international travelers returning to Europe and North America (2–9). Joint pain, the most prominent symptom, is often long-lasting (several months), sometimes incapacitating (4,6,7,9), and may recur (8). Arthralgia develops during the acute phase and symmetrically affects the wrists, ankles, and small joints of hands and feet. Joint swelling may occur initially, but permanent joint damage has not been described (5). The clinical disease and diagnostic procedures have been described (1–9), but immunologic parameters and their possible role in the clinical follow-up of patients (i.e., during the postacute long-lasting arthralgia period) remain to be investigated. n nTo further our knowledge of MAYV infection, we analyzed cytokine levels in serum samples from 6 travelers to South America who returned to Europe with Mayaro fever (MF). Two of the cases occurred during 2014; 4 occurred during 2011–2013 (2–5). n nThe 6 travelers comprised 2 men and 4 women who were 20–54 (median 36) years of age (Technical Appendix Table). The 2 most recent cases occurred in spring 2014 in a 28-year-old female student and a 54-year-old male physician. Serologic testing was performed for both patients at the Bernhard Nocht Institute and confirmed by virus neutralization testing (4). n nThe student had traveled for 3 weeks in Ecuador, visiting rainforest villages and hiking in the jungle. During her stay, she had experienced myalgia of the forearms, arthralgia of fingers and toes, subfebrile but elevated body temperatures, and maculopapular exanthema. On examination in Germany, the student had no clinical signs of disease, but she reported arthralgia of the ankles and hands. Laboratory test results showed a slightly increased C-reactive protein level (6.2 mg/L, reference value <5 mg/L); liver and kidney values and blood count were within reference ranges. MAYV indirect immunofluorescence assay showed positive IgM and IgG titers (1:320 and 1:2,560, respectively; cut-off for both was <1:20) (3). Acute MF was diagnosed. Two weeks later, follow-up serologic testing showed negative IgM but unchanged IgG titers. Arthralgia with stiffness lasted for 6 weeks. n nThe physician had traveled for 3 weeks through the jungle in Bolivia, during which time headache, myalgia, shivers, and fatigue developed, followed by foot arthralgia and maculopapular exanthema. On his return to Germany, he was seen in an outpatient practice for persisting (2 months) bilateral foot pain. Laboratory test results showed C-reactive protein levels, and liver and kidney function values, and full blood count within reference ranges. MAYV indirect immunofluorescence assay was negative for IgM but positive for IgG (1:20,480). Postacute MAYV infection was diagnosed. Two months later, follow-up serologic testing showed unchanged titers. Arthralgia with pronounced morning stiffness lasted for 6 months. n nAfter obtaining written consent from all patients, we subjected their serum samples to multiplex cytokine serum analyses (Bio-Rad Laboratories, Munich, Germany). Blood was drawn at different times after symptom onset (15–117 days). Serum samples were classified as acute ( 30 days after symptom onset, with arthralgia, n = 8). Twenty serum samples from healthy blood donors were run in parallel. During the acute phase of MF, interleukin (IL) 10, IL-12p70, RANTES (regulated on activation, normal T cell expressed and secreted), and vascular endothelial growth factor concentrations for patients were significantly elevated compared with those for healthy controls (Figure). Furthermore, a significant decrease was noted for eotaxin levels during the acute phase of disease. In the postacute arthralgic recovery phase, concentrations of IL-5–10, IL-13, IL-17, IP-10 (interferon-γ–induced protein 10), RANTES, macrophage inflammatory proteins 1α and 1β, granulocyte-macrophage colony-stimulating factor, and interferon-γ were significantly higher than those for healthy controls. TNF-α concentrations showed a nonsignificant median decrease during the acute phase (Figure). No significant changes in either phase were demonstrated for IL-1b, IL-2, IL-4, basic fibroblast growth factor, granulocyte colony-stimulating factor, monocyte chemotactic protein 1, and platelet-derived growth factor β polypeptide levels (data not shown). Cytokine levels measured in the acute phase did not differ significantly from those measured in the recovery phase. n n n nFigure n nChanges in cytokine and growth factor levels in the acute and recovery phase of Mayaro fever. Box-and-whisker plots show median, upper and lower quartile, minimum, and maximum values. A) During the prolonged recovery phase, serum levels of interleukin ... n n n nThe most striking clinical feature of MF is long-lasting arthralgia, similar to that seen in chikungunya fever, which develops in the acute phase and persists thereafter. In the travel-associated cases, arthralgia lasted for 2 to >12 months. In the examined patients, the prolonged arthralgia recovery phase was paralleled by significantly increased proinflammatory cytokine levels, indicating ongoing inflammation, probably related to arthritis. Elevated levels of RANTES and IP-10 suggest T-cell recruitment, possibly reflecting virus persistence and replication, as described for the related Chikungunya virus (10). Thus, cytokine measurements may be helpful for monitoring patient symptoms, especially when signs of arthritis (swellings and redness) and elevated standard serum inflammatory parameters are no longer present. In our study, the picture of MF cytokine elevations paralleled those described for Chikungunya virus infection (10); these elevated levels may help to elucidate the pathogenesis of MAYV-induced arthralgia. More immunology data are required to complete this evolving picture of viral arthralgia syndromes. n nTechnical Appendix: nInternational travelers who were recently diagnosed with Mayaro fever and were included in this study. n nClick here to view.(151K, pdf)

Increased Proinflammatory Cytokine Levels in Prolonged Arthralgia in Ross River Virus Infection

Ross River virus, a mosquitoborne alphavirus, causes epidemic polyarthritis in Australia and the Pacific region. We analyzed serum cytokine, chemokine, and growth factor levels in travelers returning to Germany from Australia. Serum samples showed elevated concentrations in the acute phase of the illness and, more pronounced, in the long-lasting convalescent phase.