IFN-Stimulated Gene 15 Is an Alarmin that Boosts the CTL Response via an Innate, NK Cell–Dependent Route

Type I IFN is produced upon infection and tissue damage and induces the expression of many IFN-stimulated genes (ISGs) that encode host-protective proteins. ISG15 is a ubiquitin-like molecule that can be conjugated to proteins but is also released from cells in a free form. Free, extracellular ISG15 is suggested to have an immune-regulatory role, based on disease phenotypes of ISG15-deficient humans and mice. However, the underlying mechanisms by which free ISG15 would act as a "cytokine" are unclear and much debated. We, in this study, demonstrate in a clinically relevant mouse model of therapeutic vaccination that free ISG15 is an alarmin that induces tissue alert, characterized by extracellular matrix remodeling, myeloid cell infiltration, and inflammation. Moreover, free ISG15 is a potent adjuvant for the CTL response. ISG15 produced at the vaccination site promoted the vaccine-specific CTL response by enhancing expansion, short-lived effector and effector/memory differentiation of CD8(+) T cells. The function of free ISG15 as an extracellular ligand was demonstrated, because the equivalents in murine ISG15 of 2 aa recently implicated in binding of human ISG15 to LFA-1 in vitro were required for its adjuvant effect in vivo. Moreover, in further agreement with the in vitro findings on human cells, free ISG15 boosted the CTL response in vivo via NK cells in the absence of CD4(+) T cell help. Thus, free ISG15 is part of a newly recognized innate route to promote the CTL response.Tumorimmunolog

Effects of doxycycline on heartworm embryogenesis, transmission, circulating microfilaria, and adult worms in microfilaremic dogs

Tetracycline treatment of animals or humans infected with filariae that harbor Wolbachia endosymbionts blocks further embryogenesis, and existing microfilariae gradually die. This treatment also kills developing larvae and has a slow-kill effect on adult filariae, all presumably due to elimination of the Wolbachia. Also, Dirofilaria immitis microfilariae in blood collected from dogs up to 25 days after the last dose of doxycycline developed to infective L3 that were normal in appearance and motility in mosquitoes but did not continue to develop or migrate normally after subcutaneous (SC) injection into dogs. The present study was designed to determine whether heartworm microfilariae collected at later times after treatment would regain the ability to continue normal development in a dog. The study also was expected to yield valuable data on the effects of treatment on microfilariae and antigen levels and adult worms. The study was conducted in 16 dogs as two separate replicates at different times. A total of five dogs (two in Replicate A and three in Replicate B) infected either by SC injection of L3 or intravenous transplantation of adult heartworms were given doxycycline orally at 10mg/kg twice daily for 30 days, with three untreated controls. Microfilarial counts in the five treated dogs gradually declined during the 12-13 months after treatment initiation. Two dogs were amicrofilaremic before necropsy and three had 13 or fewer microfilariae/ml. Only one treated dog was negative for heartworm antigen before necropsy. Overall, treated dogs generally had fewer live adult heartworms than controls, and most of their live worms were moribund. All three control dogs remained positive for microfilariae and antigen and had many live worms. L3 from mosquitoes fed on blood collected 73-77 or 161-164 days after initiation of doxycycline treatments were injected SC into five dogs. None of the dogs injected with L3 from mosquitoes fed on blood from doxycycline-treated dogs were ever positive for microfilariae or antigen, and none had worms at necropsy; three control dogs were positive for microfilariae and antigen and had many live worms. These data indicate that doxycycline treatment of microfilaremic dogs gradually reduces numbers of microfilariae and blocks further transmission of heartworms. This latter effect should be highly effective in reducing the rate of selection of heartworms with genes that confer resistance to macrocyclic lactone preventives and microfilaricides. The data also suggest that doxycycline has a slow-kill effect on adult heartworms

Long-term evaluation of viability of microfilariae and intravenously transplanted adult Dirofilaria immitis in microfilaremic dogs treated with low-dose, short- and long-treatment regimens of doxycycline and ivermectin

Abstract Background Microfilarial (mf) counts were monitored over 21.3 months for any rebound that might occur in counts, and adulticidal efficacy was assessed following administration of low dosage with short- and long-treatment regimens of doxycycline and ivermectin to heartworm-microfilaremic dogs. Methods Twelve heartworm-naïve beagles infected with 10 pairs of adult Dirofilaria immitis by intravenous transplantation were randomly allocated to three groups of four dogs. All treatments started on day 0. On day 0, Group 1 (short-treatment regimen) received doxycycline orally at 10 mg/kg once daily for 30 days plus ivermectin orally (minimum, 6 mcg/kg) on days 0 and 30. Group 2 (long-treatment regimen) received doxycycline orally at 10 mg/kg once daily until individual dogs became mf-negative (72–98 days) and ivermectin every other week until individual dogs became mf-negative (6–7 doses). Group 3 was the untreated control. Mf counts and antigen (Ag) tests were conducted. Dogs were necropsied for recovery and enumeration of heartworms on day 647. Results Day −1 mean mf counts were 15,613, 23,950, and 15,513 mf/ml for groups 1, 2, and 3, respectively. Mean counts for Groups 1 and 2 declined until days 239 and 97, respectively, when all were negative. Group 3 had high mf counts throughout the study. There was not a rebound in mf counts in any of the treated dogs after they became amicrofilaremic. All dogs in group 1 and group 3 were Ag-positive throughout the study and had at least one live female worm at necropsy. All dogs in treated Group 2 were positive for Ag through day 154, but were antigen-negative on days 644 and 647, as all had only male worms. Mean live adult worm recoveries for Groups 1, 2, and 3 were 6.8 (range, 5–8), 3.3 (range, 1–6), and 16.0 (range, 14–17), respectively, with a percent reduction in adult worm counts of 57.5% for Group 1 and 79.3% for Group 2. Conclusions These data lend support to the use of the American Heartworm Society Canine Guidelines for adulticide therapy recommending the initiation of doxycycline plus a macrocyclic lactone (ML) at the time of the heartworm-positive diagnosis. Graphical Abstrac

Determining the molecular drivers of species-specific interferon-stimulated gene product 15 interactions with nairovirus ovarian tumor domain proteases.

Tick-borne nairoviruses (order Bunyavirales) encode an ovarian tumor domain protease (OTU) that suppresses the innate immune response by reversing the post-translational modification of proteins by ubiquitin (Ub) and interferon-stimulated gene product 15 (ISG15). Ub is highly conserved across eukaryotes, whereas ISG15 is only present in vertebrates and shows substantial sequence diversity. Prior attempts to address the effect of ISG15 diversity on viral protein-ISG15 interactions have focused on only a single species ISG15 or a limited selection of nairovirus OTUs. To gain a more complete perspective of OTU-ISG15 interactions, we biochemically assessed the relative activities of 14 diverse nairovirus OTUs for 12 species ISG15 and found that ISG15 activity is predominantly restricted to particular nairovirus lineages reflecting, in general, known virus-host associations. To uncover the underlying molecular factors driving OTUs affinity for ISG15, X-ray crystal structures of Kupe virus and Ganjam virus OTUs bound to sheep ISG15 were solved and compared to complexes of Crimean-Congo hemorrhagic fever virus and Erve virus OTUs bound to human and mouse ISG15, respectively. Through mutational and structural analysis seven residues in ISG15 were identified that predominantly influence ISG15 species specificity among nairovirus OTUs. Additionally, OTU residues were identified that influence ISG15 preference, suggesting the potential for viral OTUs to adapt to different host ISG15s. These findings provide a foundation to further develop research methods to trace nairovirus-host relationships and delineate the full impact of ISG15 diversity on nairovirus infection

Rapid and sensitive detection of SARS-CoV-2 antibodies by biolayer interferometry.

Serological testing to evaluate antigen-specific antibodies in plasma is generally performed by rapid lateral flow test strips that lack quantitative results or by high complexity immunoassays that are time- and labor-intensive but provide semi-quantitative results. Here, we describe a novel application of biolayer interferometry for the rapid detection of antigen-specific antibody levels in plasma samples, and demonstrate its utility for quantification of SARS-CoV-2 antibodies. Our biolayer interferometry immunosorbent assay (BLI-ISA) utilizes single-use biosensors in an automated "dip-and-read" format, providing real-time optical measurements of antigen loading, plasma antibody binding, and antibody isotype detection. Complete semi-quantitative results are obtained in less than 20 min. BLI-ISA meets or exceeds the performance of high complexity methods such as Enzyme-Linked Immunosorbent Assay (ELISA) and Chemiluminescent Immunoassay. Importantly, our method can be immediately implemented on existing BLI platforms for urgent COVID-19 studies, such as serosurveillance and the evaluation of vaccine candidates. In a broader sense, BLI-ISA can be developed as a novel diagnostic platform to evaluate antibodies and other biomolecules in clinical specimens

The Domestic Dog as a Laboratory Host for Brugia malayi

Of the three nematodes responsible for lymphatic filariasis in humans, only Brugia malayi is actively maintained in research settings owing to its viability in small animal hosts, principal among which is the domestic cat. While the microfilaremic feline host is necessary for propagation of parasites on any significant scale, this system is plagued by a number of challenges not as pronounced in canine filarial models. For this reason, we investigated the capacity in which dogs may serve as competent laboratory hosts for B. malayi. We infected a total of 20 dogs by subcutaneous injection of 500 B. malayi third-stage larvae (L3) in either a single (n = 10) or repeated infection events (125 L3 per week for four weeks; n = 10). Within each group, half of the individuals were injected in the inguinal region and half in the dorsum of the hind paw. To track the course of microfilaremia in this host, blood samples were examined by microscopy biweekly for two years following infection. Additionally, to identify cellular responses with potential value as predictors of patency, we measured peripheral blood leukocyte counts for the first year of infection. A total of 10 of 20 dogs developed detectable microfilaremia. Peak microfilaria density varied but attained levels useful for parasite propagation (median = 1933 mL−1; range: 33–9950 mL−1). Nine of these dogs remained patent at 104 weeks. A two-way ANOVA revealed no significant differences between infection groups in lifetime microfilaria production (p = 0.42), nor did regression analysis reveal any likely predictive relationships to leukocyte values. The results of this study demonstrate the competence of the dog as a host for B. malayi and its potential to serve in the laboratory role currently provided by the cat, while also clarifying the potential for zoonosis in filariasis-endemic regions

Rapid and sensitive detection of SARS-CoV-2 antibodies by biolayer interferometry.

Serological testing to evaluate antigen-specific antibodies in plasma is generally performed by rapid lateral flow test strips that lack quantitative results or by high complexity immunoassays that are time- and labor-intensive but provide semi-quantitative results. Here, we describe a novel application of biolayer interferometry for the rapid detection of antigen-specific antibody levels in plasma samples, and demonstrate its utility for quantification of SARS-CoV-2 antibodies. Our biolayer interferometry immunosorbent assay (BLI-ISA) utilizes single-use biosensors in an automated "dip-and-read" format, providing real-time optical measurements of antigen loading, plasma antibody binding, and antibody isotype detection. Complete semi-quantitative results are obtained in less than 20 min. BLI-ISA meets or exceeds the performance of high complexity methods such as Enzyme-Linked Immunosorbent Assay (ELISA) and Chemiluminescent Immunoassay. Importantly, our method can be immediately implemented on existing BLI platforms for urgent COVID-19 studies, such as serosurveillance and the evaluation of vaccine candidates. In a broader sense, BLI-ISA can be developed as a novel diagnostic platform to evaluate antibodies and other biomolecules in clinical specimens

Probing the impact of nairovirus genomic diversity on viral ovarian tumor domain protease (vOTU) structure and deubiquitinase activity.

Post-translational modification of host and viral proteins by ubiquitin (Ub) and Ub-like proteins, such as interferon stimulated gene product 15 (ISG15), plays a key role in response to infection. Viruses have been increasingly identified that contain proteases possessing deubiquitinase (DUB) and/or deISGylase functions. This includes viruses in the Nairoviridae family that encode a viral homologue of the ovarian tumor protease (vOTU). vOTU activity was recently demonstrated to be critical for replication of the often-fatal Crimean-Congo hemorrhagic fever virus, with DUB activity suppressing the type I interferon responses and deISGylase activity broadly removing ISG15 conjugated proteins. There are currently about 40 known nairoviruses classified into fourteen species. Recent genomic characterization has revealed a high degree of diversity, with vOTUs showing less than 25% amino acids identities within the family. Previous investigations have been limited to only a few closely related nairoviruses, leaving it unclear what impact this diversity has on vOTU function. To probe the effects of vOTU diversity on enzyme activity and specificity, we assessed representative vOTUs spanning the Nairoviridae family towards Ub and ISG15 fluorogenic substrates. This revealed great variation in enzymatic activity and specific substrate preferences. A subset of the vOTUs were further assayed against eight biologically relevant di-Ub substrates, uncovering both common trends and distinct preferences of poly-Ub linkages by vOTUs. Four novel X-ray crystal structures were obtained that provide a biochemical rationale for vOTU substrate preferences and elucidate structural features that distinguish the vOTUs, including a motif in the Hughes orthonairovirus species that has not been previously observed in OTU domains. Additionally, structure-informed mutagenesis provided the first direct evidence of a second site involved in di-Ub binding for vOTUs. These results provide new insight into nairovirus evolution and pathogenesis, and further enhances the development of tools for therapeutic purposes