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Truncated reverse transcription of HIV RNA produces a single-stranded DNA intermediate with a unique Y-DNA stem-loop structure flanked by unpaired guanines. Schlee and colleagues show this Y-DNA activates cGAS to elicit the production of type I interferon. Cytosolic DNA that emerges during infection with a retrovirus or DNA virus triggers antiviral type I interferon responses. So far, only double-stranded DNA (dsDNA) over 40 base pairs (bp) in length has been considered immunostimulatory. Here we found that unpaired DNA nucleotides flanking short base-paired DNA stretches, as in stem-loop structures of single-stranded DNA (ssDNA) derived from human immunodeficiency virus type 1 (HIV-1), activated the type I interferon–inducing DNA sensor cGAS in a sequence-dependent manner. DNA structures containing unpaired guanosines flanking short (12- to 20-bp) dsDNA (Y-form DNA) were highly stimulatory and specifically enhanced the enzymatic activity of cGAS. Furthermore, we found that primary HIV-1 reverse transcripts represented the predominant viral cytosolic DNA species during early infection of macrophages and that these ssDNAs were highly immunostimulatory. Collectively, our study identifies unpaired guanosines in Y-form DNA as a highly active, minimal cGAS recognition motif that enables detection of HIV-1 ssDNA.

High-throughput immune repertoire sequencing is promising to lead to new statistical diagnostic tools for medicine and biology. Successful implementations of these methods require a correct characterization, analysis, and interpretation of these data sets. We present IGoR (Inference and Generation Of Repertoires)—a comprehensive tool that takes B or T cell receptor sequence reads and quantitatively characterizes the statistics of receptor generation from both cDNA and gDNA. It probabilistically annotates sequences and its modular structure can be used to investigate models of increasing biological complexity for different organisms. For B cells, IGoR returns the hypermutation statistics, which we use to reveal co-localization of hypermutations along the sequence. We demonstrate that IGoR outperforms existing tools in accuracy and estimate the sample sizes needed for reliable repertoire characterization. B and T cell receptor diversity can be studied by high-throughput immune receptor sequencing. Here, the authors develop a software tool, IGoR, that calculates the likelihoods of potential V(D)J recombination and somatic hypermutation scenarios from raw immune sequence reads.

Inappropriate or chronic detection of self nucleic acids by the innate immune system underlies many human autoimmune diseases. We discuss here an unexpected source of endogenous immunostimulatory nucleic acids: the reverse-transcribed cDNA of endogenous retroelements. The interplay between innate immune sensing and clearance of retroelement cDNA has important implications for the understanding of immune responses to infectious retroviruses such as human immunodeficiency virus (HIV). Furthermore, the detection of cDNA by the innate immune system reveals an evolutionary tradeoff: selection for a vigorous, sensitive response to infectious retroviruses may predispose the inappropriate detection of endogenous retroelements. We propose that this tradeoff has placed unique constraints on the sensitivity of the DNA-activated antiviral response, with implications for the interactions of DNA viruses and retroviruses with their hosts. Finally, we discuss how better understanding of the intersection of retroelement biology and innate immunity can guide the way to novel therapies for specific autoimmune diseases.

Human immunodeficiency virus type 1 (HIV-1) is known to activate cytosolic DNA sensor pathways, such as the cGAS-STING pathway, the activation of which leads to interferon production. The primary source of this activation is reverse transcription of the viral RNA genome into cDNA, which occurs in the cytoplasm. However, the degree of cytosolic DNA sensor activation during HIV-1 infection is significantly lower compared to that induced by DNA-containing viruses or even by the related HIV-2. This can be attributed to the successful evasion of innate immune recognition pathways by HIV-1, particularly through the disruption of cGAS-STING signaling pathway activation. In this review, we summarize the available information regarding the mechanisms employed by HIV-1 to conceal viral cDNA from cytosolic DNA sensors. Deciphering these mechanisms may reveal potential vulnerabilities that could be targeted to develop novel antiviral approaches.

The Toll pathway is one of the most important signaling pathways regulating insect innate immunity. Spatzle is a key protein that functions as a Toll receptor ligand to trigger Toll-dependent expression of immunity-related genes. In this study, a novel spatzle gene (ApSPZ) from the Chinese oak silkworm Antheraea pernyi was identified. The ApSPZ cDNA is 1065 nucleotides with an open reading frame (ORF) of 777 bp encoding a protein of 258 amino acids. The protein has an estimated molecular weight of 29.71 kDa and an isoelectric point (PI) of 8.53. ApSPZ is a nuclear and secretory protein with no conserved domains or membrane helices and shares 40% amino acid identity with SPZ from Manduca sexta. Phylogenetic analysis indicated that ApSPZ might be a new member of the Spatzle type 1 family, which belongs to the Spatzle superfamily. The expression patterns of several genes involved in the Toll pathway were examined at different developmental stages and various tissues in 5th instar larvae. The examined targets included A. pernyi spatzle, GNBP, MyD88, Tolloid, cactus and dorsalA. The RT-PCR results showed that these genes were predominantly expressed in immune-responsive fat body tissue, indicating that the genes play a crucial role in A. pernyi innate immunity. Moreover, A. pernyi infection with the fungus Nosema pernyi and the gram-positive bacterium Enterococcus pernyi, but not the gram-negative bacterium Escherichia coli, activated the Toll signaling pathway. These results represent the first study of the Toll pathway in A. pernyi, which provides insight into the A. pernyi innate immune system.

Zika virus (ZIKV) is a mosquito-borne member of the Flaviviridae family that has been known to circulate for decades causing mild febrile illness. The more recent ZIKV outbreaks in the Americas and the Caribbean associated with congenital malformations and Guillain-Barré syndrome in adults have placed public health officials in high alert and highlight the significant impact of ZIKV on human health. New technologies to study the biology of ZIKV and to develop more effective prevention options are highly desired. In this study we demonstrate that direct delivery in mice of an infectious ZIKV cDNA clone allows the rescue of recombinant (r)ZIKV in vivo . A bacterial artificial chromosome containing the sequence of ZIKV strain Paraiba/2015 under the control of the cytomegalovirus promoter was complexed with a commercial transfection reagent and administrated using different routes in type-I interferon receptor deficient A129 mice. Clinical signs and death associated with ZIKV viremia were observed in mice. The rZIKV recovered from these mice remained fully virulent in a second passage in mice. Interestingly, infectious rZIKV was also recovered after intraperitoneal inoculation of the rZIKV cDNA in the absence of transfection reagent. Further expanding these studies, we demonstrate that a single intraperitoneal inoculation of a cDNA clone encoding an attenuated rZIKV was safe, highly immunogenic, and provided full protection against lethal ZIKV challenge. This novel in vivo reverse genetics method is a potentially suitable delivery platform for the study of wild-type and live-attenuated ZIKV devoid of confounding factors typical associated with in vitro systems. Moreover, our results open the possibility of employing similar in vivo reverse genetic approaches for the generation of other viruses and, therefore, change the way we will use reverse genetics in the future.

Trichinellosis has major economic impacts on animal husbandry and food safety, and the control and elimination of trichinellosis is a major objective of veterinary medicine. A gene encoding serine protease of Trichinella spiralis (Ts-Adsp) was identified by immunoscreening an adult T. spiralis cDNA library. In this study, the recombinant Ts-Adsp protein (rTs-Adsp) was cloned and expressed in a prokaryotic expression system and purified by Ni-affinity chromatography. To determine whether the purified rTs-Adsp is a potential vaccine candidate for the control of T. spiralis infection, we immunized BALB/c mice with this protein in combination with an alum adjuvant and subsequently challenged with T. spiralis larvae. The results showed that mice vaccinated with rTs-Adsp exhibited an average reduction in the muscle larvae burden of 46.5% relative to the control group. Immunization with the rTs-Adsp antigen induced both humoral and cellular immune responses, which manifested as elevated specific anti–rTs-Adsp IgG and IgE antibodies and a mixed Th1–Th2 response, as determined by Th1 (IFN-γ and IL-2) and Th2 (IL-4, IL-10, and IL-13) cytokine profiling, with the Th2 predominant. Thus, purified rTs-Adsp is able to limit the invasion of T. spiralis, and this protein could be an effective vaccine candidate for trichinellosis.

Schizothorax prenanti (S. prenanti) is mainly distributed in the upstream regions of the Yangtze River and its tributaries in China. This species is indigenous and commercially important. However, in recent years, wild populations and aquacultures have faced the serious challenges of germplasm variation loss and an increased susceptibility to a range of pathogens. Currently, the genetics and immune mechanisms of S. prenanti are unknown, partly due to a lack of genome and transcriptome information. Here, we sought to identify genes related to immune functions and to identify molecular markers to study the function of these genes and for trait mapping. To this end, the transcriptome from spleen tissues of S. prenanti was analyzed and sequenced. Using paired-end reads from the Illumina Hiseq2500 platform, 48,517 transcripts were isolated from the spleen transcriptome. These transcripts could be clustered into 37,785 unigenes with an N50 length of 2,539 bp. The majority of the unigenes (35,653, 94.4%) were successfully annotated using non-redundant nucleotide sequence analysis (nt), and the non-redundant protein (nr), Swiss-Prot, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. KEGG pathway assignment identified more than 500 immune-related genes. Furthermore, 7,545 putative simple sequence repeats (SSRs), 857,535 single nucleotide polymorphisms (SNPs), and 53,481 insertion/deletion (InDels) were detected from the transcriptome. This is the first reported high-throughput transcriptome analysis of S. prenanti, and it provides valuable genetic resources for the investigation of immune mechanisms, conservation of germplasm, and molecular marker-assisted breeding of S. prenanti.

Background: Insulin-like growth factor-binding protein 7 (IGFBP7) is an abundant, selective and accessible biomarker of glioblastoma multiforme (GBM) tumour vessels. In this study, an anti-IGFBP7 single-domain antibody (sdAb) was developed to target GBM vessels for molecular imaging applications. Methods: Human GBM was modelled in mice by intracranial implantation of U87MG.EGFRvIII cells. An anti-IGFBP7 sdAb, isolated from an immune llama library by panning, was assessed in vitro for its binding affinity using surface plasmon resonance and by ex vivo immunobinding on mouse and human GBM tissue. Tumour targeting by Cy5.5-labelled anti-IGFBP7 sdAb as well as by anti-IGFBP7 sdAb conjugated to PEGylated Fe 3 O 4 nanoparticles (NPs)-Cy5.5 were assessed in U87MG.EGFRvIII tumour-bearing mice in vivo using optical imaging and in brain sections using fluorescent microscopy. Results: Surface plasmon resonance analyses revealed a medium affinity ( K D =40–50 n M ) binding of the anti-IGFBP7 sdAb to the purified antigen. The anti-IGFBP7 sdAb also selectively bound to both mouse and human GBM vessels, but not normal brain vessels in tissue sections. In vivo , intravenously injected anti-IGFBP7 sdAb-Cy5.5 bound to GBM vessels creating high imaging signal in the intracranial tumour. Similarly, the anti-IGFBP7 sdAb-functionalised PEGylated Fe 3 O 4 NP-Cy5.5 demonstrated enhanced tumour signal compared with non-targeted NPs. Fluorescent microscopy confirmed the presence of anti-IGFBP7 sdAb and anti-IGFBP7 sdAb-PEGylated Fe 3 O 4 NPs selectively in GBM vessels. Conclusions: Anti-IGFBP7 sdAbs are novel GBM vessel-targeting moieties suitable for molecular imaging.

We have generated polyclonal and monoclonal antibodies by genetic immunization over the last two decades. In this paper, we present our most successful methodology acquired over these years and present the animals in which we obtained the highest rates of success. The technique presented is convenient, easy, affordable, and generates antibodies against mammalian proteins in their native form. This protocol requires neither expensive equipment, such as a gene gun, nor sophisticated techniques such as the conjugation of gold microspheres, electroporation, or surgery to inject in lymph nodes. The protocol presented uses simply the purified plasmid expressing the protein of interest under a strong promoter, which is injected at intramuscular and intradermal sites. This technique was tested in five species. Guinea pigs were the animals of choice for the production of polyclonal antibodies. Monoclonal antibodies could be generated in mice by giving, as a last injection, a suspension of transfected cells. The antibodies detected their antigens in their native forms. They were highly specific with very low non-specific background levels, as assessed by immune-blots, immunocytochemistry, immunohistochemistry and flow cytometry. We present herein a detailed and simple procedure to successfully raise specific antibodies against native proteins.