CCR5 serves as an essential coreceptor for human immunodeficiency virus type 1 (HIV-1) entry, and individuals with a CCR5 D32 variant appear to be healthy, making CCR5 an attractive target for control of HIV-1 infection. The CRISPR/Cas9, which functions as a naturally existing adaptive immune system in prokaryotes, has been recently harnessed as a novel nuclease system for genome editing in mammalian cells. Although CRISPR/Cas9 can be readily delivered into cell lines, due to the large size of the Cas9 protein, efficient delivery of CCR5-targeting CRISPR/Cas9 components into primary cells, including CD4 + T-cells, the primary target for HIV-1 infection in vivo, remains a challenge. In the current study, following design of a panel of top-ranked single-guided RNAs (sgRNAs) targeting the ORF of CCR5, we demonstrate that CRISPR/Cas9 can efficiently mediate the editing of the CCR5 locus in cell lines, resulting in the knockout of CCR5 expression on the cell surface. Next-generation sequencing revealed that various mutations were introduced around the predicted cleavage site of CCR5. For each of the three most effective sgRNAs that we analysed, no significant off-target effects were detected at the 15 top-scoring potential sites. More importantly, by constructing chimeric Ad5F35 adenoviruses carrying CRISPR/Cas9 components, we efficiently transduced primary CD4 + Tlymphocytes and disrupted CCR5 expression, and the positively transduced cells were conferred with HIV-1 resistance. To our knowledge, this is the first study establishing HIV-1 resistance in primary CD4 + T-cells utilizing adenovirus-delivered CRISPR/Cas9.

SummaryToll-like receptor-3 (TLR-3) recognizes double-stranded RNA and induces multiple intracellular events responsible for innate anti-viral immunity against a number of viral infections. Activation of TLR-3 inhibits human immunodeficiency virus (HIV) replication, but the mechanism(s) underlying the action of TLR-3 activation on HIV are largely unknown. Here we demonstrate that treatment of monocyte-derived macrophages with poly I:C, a synthetic ligand for TLR-3, significantly inhibited HIV infection and replication. Investigation of the mechanisms showed that TLR-3 activation resulted in the induction of type I interferon inducible antiviral factors, including APOBEC3G and tetherin, the newly identified anti-HIV cellular proteins. In addition, poly I:C-treated macrophages expressed increased levels of CC chemokines, the ligands for CCR5. Furthermore, TLR-3 activation in macrophages induced the expression of cellular microRNAs , the newly identified intracellular HIV restriction factors. These findings indicate that TLR-3-mediated induction of multiple anti-HIV factors should be beneficial for the treatment of HIV disease where innate immune responses are compromised by the virus.

Chemokine (C–C motif) ligand 19 (CCL19) is a critical regulator of the induction of T cell activation, immune tolerance, and inflammatory responses during continuous immune surveillance, homeostasis, and development. Migration of CC-chemokine receptor 7 (CCR7)-expressing cells to secondary lymphoid organs is a crucial step in the onset of adaptive immunity, which is initiated by a complex interaction between CCR7 and its cognate ligands. Recent advances in knowledge regarding the response of the CCL19-CCR7 axis to viral infections have elucidated the complex network of interplay among the invading virus, target cells and host immune responses. Viruses use various strategies to evade or delay the cytokine response, gaining additional time to replicate in the host. In this review, we summarize the impacts of CCL19 and CCR7 expression on the regulation of viral pathogenesis with an emphasis on the corresponding signaling pathways and adjuvant mechanisms. We present and discuss the expression, signaling adaptor proteins and effects of CCL19 and CCR7 as these molecules differentially impact different viral infections and viral life cycles in host homeostatic strategies. The underlying mechanisms discussed in this review may assist in the design of novel agents to modulate chemokine activity for viral prevention.

L-ficolin, one of lectin families, is a recently identified complement factor that initiates lectin pathway of complement. Little is known about its role in viral hepatitis. In the present study, we found that L-ficolin in serum from 103 patients with hepatitis C virus (HCV), were significantly higher than that in 150 healthy controls. We further found that L-ficolin expressions were significantly increased in vitro study by HCV JFH-1 infected human hepatocyte cell line Huh7.5.

Summary Nasal administration has emerged as a promising and attractive route for vaccination, especially for the prophylaxis of respiratory diseases. Our previous studies have shown that severe acute respiratory syndrome coronavirus (SARS‐CoV) virus‐like particles (VLPs) can be assembled using a recombinant baculovirus (rBV) expression system and such VLPs induce specific humoral and cellular immune responses in mice after subcutaneous injection. Here, we investigated mucosal immune responses to SARS‐CoV VLPs in a mouse model. Mice were immunized in parallel, intraperitoneally or intranasally, with VLPs alone or with VLPs plus cytosine–phosphate–guanosine (CpG). Immune responses, including the production of SARS‐CoV‐specific serum immunoglobulin G (IgG) and secretory immunoglobulin A (sIgA), were determined in mucosal secretions and tissues. Both immunizations induced SARS‐CoV‐specific IgG, although the levels of IgG in groups immunized via the intraperitoneal (i.p.) route were higher. sIgA was detected in saliva in groups immunized intranasally but not in groups immunized intraperitoneally. CpG had an adjuvant effect on IgA production in genital tract washes when administered intranasally but only affected IgA production in faeces samples when administered intraperitoneally. In addition, IgA was also detected in mucosal tissues from the lung and intestine, while CpG induced an increased level of IgA in the intestine. Most importantly, neutralization antibodies were detected in sera after i.p. and intranasal (i.n.) immunizations. Secretions in genital tract washes from the i.n. group also showed neutralization activity. Furthermore, VLPs that were administered intraperitoneally elicited cellular immune responses as demonstrated by enzyme‐linked immunospot (ELISPOT) assay analyses. In summary, our study indicates that mucosal immunization with rBV SARS‐CoV VLPs represent an effective means for eliciting protective systemic and mucosal immune responses against SARS‐CoV, providing important information for vaccine design.

Highlights d We describe a heterotrimeric configuration of Nb-Nb H -Nb (Nb 15 -Nb H -Nb 15 ) d Nb 15 -Nb H -Nb 15 exhibits improved viral inhibition and half-life d Nb 15 -Nb H -Nb 15 provides ultrapotent neutralization against variants Alpha and Delta d Intranasal delivery of Nbs protects SARS-CoV-2 infection in hACE2 transgenic mice

BackgroundMany viruses depend on the extensive membranous network of the endoplasmic reticulum (ER) for their translation, replication, and packaging. Certain membrane modifications of the ER can be a trigger for ER stress, as well as the accumulation of viral protein in the ER by viral infection. Then, unfolded protein response (UPR) is activated to alleviate the stress. Zika virus (ZIKV) is a mosquito-borne flavivirus and its infection causes microcephaly in newborns and serious neurological complications in adults. Here, we investigated ER stress and the regulating model of UPR in ZIKV-infected neural cells in vitro and in vivo.MethodsMice deficient in type I and II IFN receptors were infected with ZIKV via intraperitoneal injection and the nervous tissues of the mice were assayed at 5 days post-infection. The expression of phospho-IRE1, XBP1, and ATF6 which were the key markers of ER stress were analyzed by immunohistochemistry assay in vivo. Additionally, the nuclear localization of XBP1s and ATF6n were analyzed by immunohistofluorescence. Furthermore, two representative neural cells, neuroblastoma cell line (SK-N-SH) and astrocytoma cell line (CCF-STTG1), were selected to verify the ER stress in vitro. The expression of BIP, phospho-elF2α, phospho-IRE1, and ATF6 were analyzed through western blot and the nuclear localization of XBP1s was performed by confocal immunofluorescence microscopy. RT-qPCR was also used to quantify the mRNA level of the UPR downstream genes in vitro and in vivo.ResultsZIKV infection significantly upregulated the expression of ER stress markers in vitro and in vivo. Phospho-IRE1 and XBP1 expression significantly increased in the cerebellum and mesocephalon, while ATF6 expression significantly increased in the mesocephalon. ATF6n and XBP1s were translocated into the cell nucleus. The levels of BIP, ATF6, phospho-elf2α, and spliced xbp1 also significantly increased in vitro. Furthermore, the downstream genes of UPR were detected to investigate the regulating model of the UPR during ZIKV infection in vitro and in vivo. The transcriptional levels of atf4, gadd34, chop, and edem-1 in vivo and that of gadd34 and chop in vitro significantly increased.ConclusionFindings in this study demonstrated that ZIKV infection activates ER stress in neural cells. The results offer clues to further study the mechanism of neuropathogenesis caused by ZIKV infection.Electronic supplementary materialThe online version of this article (10.1186/s12974-018-1311-5) contains supplementary material, which is available to authorized users.

Induction of broad and potent neutralizing Abs at the mucosal portals of entry remains a primary goal for most vaccines against mucosally acquired viral infections. Selection of appropriate adjuvants capable of promoting both systemic and mucosal responses will be crucial for the development of effective immunization strategies. In this study, we investigated whether plasmid codelivery of cytokines APRIL, CCL19, or CCL28 can enhance Ag-induced immune responses to HIV-1 gp140. Our results demonstrated that pCCL19 and pCCL28, but not pAPRIL, significantly enhanced Ag-specific systemic and mucosal Ab responses. gp140-specific Abs in serum enhanced by pCCL19 or pCCL28 were broadly distributed across all four IgG subclasses, of which IgG1 was predominant. The enhanced systemic and mucosal Abs showed increased neutralizing activity against both homologous and heterologous HIV-1, and potency correlated with gp140-specific serum IgG and vaginal IgA levels. Measurement of gp140-specific cytokines produced by splenocytes demonstrated that pCCL19 and pCCL28 augmented balanced Th1/Th2 responses. pCCL19 and pCCL28 also increased IgA+ cells in colorectal mucosal tissue. pCCL19 codelivery resulted in an increase of CCR7+ CD11c+ cells in mesenteric lymph nodes and both CCR7+ CD11c+ cells and CCR7+ CD3e+ cells in spleen, whereas pCCL28 codelivery resulted in an augment of CCR10+ CD19+ cells in both spleen and mesenteric lymph nodes. Together, our data indicate that pCCL19 and pCCL28 can enhance HIV-1 envelope–specific systemic and mucosal Ab responses, as well as T cell responses. Such enhancements appear to be associated with mobilization of responsive immunocytes into secondary lymphoid organs and mucosal tissues through interactions with corresponding receptors.