Generation of therapeutic antisera for emerging viral infections

The recent Ebola virus outbreak has highlighted the therapeutic potential of antisera and renewed interest in this treatment approach. While human convalescent sera may not be readily available in the early stages of an outbreak, antisera of animal origin can be produced in a short time frame. Here, we compared adjuvanted virus-like particles (VLP) with recombinant modified vaccinia virus Ankara and vesicular stomatitis virus (VSV), both expressing the Ebola virus antigens. The neutralizing antibody titers of rabbits immunized with adjuvanted VLPs were similar to those immunized with the replication-competent VSV, indicating that presentation of the antigen in its native conformation rather than de novo antigen expression is essential for production of functional antibodies. This approach also yielded high-titer antisera against Nipah virus glycoproteins, illustrating that it is transferable to other virus families. Multiple-step immunoglobulin G purification using a two-step 20-40% ammonium sulfate precipitation followed by protein A affinity chromatography resulted in 90% recovery of functionality and sustained in vivo stability. Adjuvanted VLP-based immunization strategies are thus a promising approach for the rapid generation of therapeutic antisera against emerging infections

Canine Distemper Viruses Expressing a Hemagglutinin without N-Glycans Lose Virulence but Retain Immunosuppression▿

Paramyxovirus glycoproteins are posttranslationally modified by the addition of N-linked glycans, which are often necessary for correct folding, processing, and cell surface expression. To establish the contribution of N glycosylation to morbillivirus attachment (H) protein function and overall virulence, we first determined the use of the potential N-glycosylation sites in the canine distemper virus (CDV) H proteins. Biochemical characterization revealed that the three sites conserved in all strains were N glycosylated, whereas only two of the up to five additional sites present in wild-type strains are used. A wild-type virus with an H protein reproducing the vaccine strain N-glycosylation pattern remained lethal in ferrets but with a prolonged course of disease. In contrast, introduction of the vaccine H protein in the wild-type context resulted in complete attenuation. To further characterize the role of N glycosylation in CDV pathogenesis, the N-glycosylation sites of wild-type H proteins were successively deleted, including a nonstandard site, to ultimately generate a nonglycosylated H protein. Despite reduced expression levels, this protein remained fully functional. Recombinant viruses expressing N-glycan-deficient H proteins no longer caused disease, even though their immunosuppressive capacities were retained, indicating that reduced N glycosylation contributes to attenuation without affecting immunosuppression

Comparative pathogenesis of Alkhumra hemorrhagic fever and Kyasanur forest disease viruses in a mouse model.

Kyasanur Forest disease virus (KFDV) and Alkhumra hemorrhagic fever virus (AHFV) are genetically closely-related, tick-borne flaviviruses that cause severe, often fatal disease in humans. Flaviviruses in the tick-borne encephalitis (TBE) complex typically cause neurological disease in humans whereas patients infected with KFDV and AHFV predominately present with hemorrhagic fever. A small animal model for KFDV and AHFV to study the pathogenesis and evaluate countermeasures has been lacking mostly due to the need of a high biocontainment laboratory to work with the viruses. To evaluate the utility of an existing mouse model for tick-borne flavivirus pathogenesis, we performed serial sacrifice studies in BALB/c mice infected with either KFDV strain P9605 or AHFV strain Zaki-1. Strikingly, infection with KFDV was completely lethal in mice, while AHFV caused no clinical signs of disease and no animals succumbed to infection. KFDV and high levels of pro-inflammatory cytokines were detected in the brain at later time points, but no virus was found in visceral organs; conversely, AHFV Zaki-1 and elevated levels of cytokines were found in the visceral organs at earlier time points, but were not detected in the brain. While infection with either virus caused a generalized leukopenia, only AHFV Zaki-1 induced hematologic abnormalities in infected animals. Our data suggest that KFDV P9605 may have lost its ability to cause hemorrhagic disease as the result of multiple passages in suckling mouse brains. However, likely by virtue of fewer mouse passages, AHFV Zaki-1 has retained the ability to replicate in visceral organs, cause hematologic abnormalities, and induce pro-inflammatory cytokines without causing overt disease. Given these striking differences, the use of inbred mice and the virus passage history need to be carefully considered in the interpretation of animal studies using these viruses

Nectin-4 Interactions Govern Measles Virus Virulence in a New Model of Pathogenesis, the Squirrel Monkey (Saimiri sciureus)

International audienceIn addition to humans, only certain nonhuman primates are naturally susceptible to measles virus (MeV) infection. Disease severity is species dependent, ranging from mild to moderate for macaques to severe and even lethal for certain New World monkey species. To investigate if squirrel monkeys (Saimiri sciureus), which are reported to develop a course of disease similar to humans, may be better suited than macaques for the identification of virulence determinants or the evaluation of therapeutics, we infected them with a green fluorescent protein-expressing MeV. Compared to cynomolgus macaques (Macaca fascicularis) infected with the same virus, the squirrel monkeys developed more-severe immunosuppression, higher viral load, and a broader range of clinical signs typical for measles. In contrast, infection with an MeV unable to interact with the epithelial receptor nectin-4, while causing immunosuppression, resulted in only a mild and transient rash and a short-lived elevation of the body temperature. Similar titers of the wild-type and nectin-4-blind MeV were detected in peripheral blood mononuclear cells and lymph node homogenates, but only the wild-type virus was found in tracheal lavage fluids and urine. Thus, our study demonstrates the importance of MeV interactions with nectin-4 for clinical disease in the new and better-performing S. sciureus model of measles pathogenesis.IMPORTANCE The characterization of mechanisms underlying measles virus clinical disease has been hampered by the lack of an animal model that reproduces the course of disease seen in human patients. Here, we report that infection of squirrel monkeys (Saimiri sciureus) fulfills these requirements. Comparative infection with wild-type and epithelial cell receptor-blind viruses demonstrated the importance of epithelial cell infection for clinical disease, highlighting the spread to epithelia as an attractive target for therapeutic strategies

Nectin-4-dependent measles virus spread to the cynomolgus monkey tracheal epithelium: role of infected immune cells infiltrating the lamina propria.

International audienceAfter the contagion measles virus (MV) crosses the respiratory epithelium within myeloid cells that express the primary receptor signaling lymphocytic activation molecule (SLAM), it replicates briskly in SLAM-expressing cells in lymphatic organs. Later, the infection spreads to epithelia expressing nectin-4, an adherens junction protein expressed preferentially in the trachea, but how it gets there is not understood. To characterize the mechanisms of spread, we infected groups of 5 or 6 cynomolgus monkeys (Macaca fascicularis) with either a wild-type MV or its "N4-blind" derivative, which is unable to enter nectin-4-expressing cells because of the targeted mutation of two hemagglutinin residues. As expected, both viruses caused similar levels of immunosuppression, as monitored by reductions in white blood cell counts and lymphocyte proliferation activity. However, monkeys infected with the N4-blind MV cleared infection more rapidly. Wild-type virus-infected monkeys secreted virus, while marginal virus titers were detected in tracheal lavage fluid cells of N4-blind MV-infected hosts. Analyses of tracheal rings obtained at necropsy (day 12) documented widespread infection of individual cells or small cell clusters in the subepithelial lamina propria of monkeys infected with either virus. However, only wild-type MV spread to the epithelium, forming numerous infectious centers comprised of many contiguous columnar cells. Infected CD11c(+) myeloid (macrophage or dendritic) cells were frequently observed in the lamina propria below epithelial infectious centers. Thus, MV may use myeloid cells as vehicles not only immediately after contagion but also to infect epithelia of tissues expressing nectin-4, including the trachea

Hematologic abnormalities induced by KFDV P9605 and AHFV Zaki-1 infection.

<p>(A) Total RBC counts from whole blood. (B) Hemoglobin (Hb) levels from whole blood. (C) Hematocrit levels from whole blood. (D) Platelet counts from whole blood. Each symbol represents one animal, and arithmetic means are indicated by horizontal black lines.</p

Virulence of KFDV and AHFV in mice.

<p>Groups of 5 mice were infected via footpad inoculation with 2×10³ TCID₅₀ per animal of either KFDV P9605, AHFV 2003, or AHFV Zaki-1. (A) Survival curve. Death of an animal is indicated by a step down on the curve. (B) Temperature. Mice were implanted with subdermal transponders as detailed in the Materials and Methods, and the temperature was monitored daily. (C) Weight change. Mice were weighed daily, and the change in weight is shown as a percentage of the initial study weight on the day of infection. Error bars in (B) and (C) represent standard deviations.</p

Differential cytokine induction by KFDV P9605 and AHFV Zaki-1.

<p>Organ homogenates were analyzed by Bio-Plex Pro Mouse Cytokine Assay as described in the Materials and Methods. Results for IL-6, IL-10, IFN-γ, MCP-1, and TNF-α are shown for the brain (A), kidney (B), and spleen (C). All cytokine concentrations were normalized to the amount of the respective tissue that was homogenized in each sample. Error bars represent standard deviations. Statistical significance is indicated by <b>*</b> for <i>p</i><0.05 and <b>**</b> for <i>p</i><0.01.</p

White blood cell (WBC) abnormalities induced by KFDV P9605 and AHFV Zaki-1 infection.

<p>(A) Total WBC counts from whole blood. Each symbol represents one animal. (B) Lymphocyte counts from whole blood. Each symbol represents one animal. Arithmetic means in (A) and (B) are indicated by horizontal black lines. (C) Composite graph showing average absolute counts of lymphocytes, neutrophils, monocytes, eosinophils, and basophils from 5 mice per day for each virus, and 10 uninfected control animals. (D) Composite graph showing average population proportions for lymphocytes, neutrophils, monocytes, eosinophils, and basophils from 5 mice per day for each virus, and 10 uninfected control animals.</p