Highly sensitive and rapid point-of-care testing for HIV-1 infection based on CRISPR-Cas13a system.

BACKGROUND: Human immunodeficiency virus type one (HIV-1) is the leading cause of acquired immunodeficiency syndrome (AIDS). AIDS remains a global public health concern but can be effectively suppressed by life-long administration of combination antiretroviral therapy. Early detection and diagnosis are two key strategies for the prevention and control of HIV/AIDS. Rapid and accurate point-of-care testing (POCT) provides critical tools for managing HIV-1 epidemic in high-risk areas and populations. METHODS: In this study, a POCT for HIV-1 RNA was developed by CRISPR-Cas13a lateral flow strip combined with reverse transcriptase recombinase-aided amplification (RT-RAA) technology, the results can be directly observed by naked eyes. RESULTS: Moreover, with the degenerate base-binding CRISPR-Cas13a system was introduced into the RT-RAA primer designing, the technology developed in this study can be used to test majority of HIV-1 RNA with limit of detection (LOD) 1 copy/µL, while no obvious cross-reaction with other pathogens. We evaluated this method for detecting HIV-1 RNA of clinical samples, the results showed that the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy were 91.81% (85.03- 96.19%), 100% (92.60-100%), 100% (96.41-100%), 39.14% (25.59-54.60%) and 92.22% (86.89-95.88%), respectively. The lowest viral load detectable by this method was 112copies/mL. CONCLUSION: Above all, this method provides a point-of-care detection of HIV-1 RNA, which is stable, simple and with good sensitivity and specificity. This method has potential to be developed for promoting early diagnosis and treatment effect monitoring of HIV patients in clinical.

Potent immunogenicity and broad-spectrum protection potential of microneedle array patch-based COVID-19 DNA vaccine candidates encoding dimeric RBD chimera of SARS-CoV and SARS-CoV-2 variants.

Breakthrough infections by SARS-CoV-2 variants pose a global challenge to COVID-19 pandemic control, and the development of more effective vaccines of broad-spectrum protection is needed. In this study, we constructed pVAX1-based plasmids encoding receptor-binding domain (RBD) chimera of SARS-CoV-1 and SARS-CoV-2 variants, including pAD1002 (encoding RBDSARS/BA1), pAD1003 (encoding RBDSARS/Beta) and pAD131 (encoding RBDBA1/Beta). Plasmids pAD1002 and pAD131 were far more immunogenic than pAD1003 in terms of eliciting RBD-specific IgG when intramuscularly administered without electroporation. Furthermore, dissolvable microneedle array patches (MAP) greatly enhanced the immunogenicity of these DNA constructs in mice and rabbits. MAP laden with pAD1002 (MAP-1002) significantly outperformed inactivated SARS-CoV-2 virus vaccine in inducing RBD-specific IFN-γ+ effector and memory T cells, and generated T lymphocytes of different homing patterns compared to that induced by electroporated DNA in mice. In consistence with the high titer neutralization results of MAP-1002 antisera against SARS-CoV-2 pseudoviruses, MAP-1002 protected human ACE2-transgenic mice from Omicron BA.1 challenge. Collectively, MAP-based DNA constructs encoding chimeric RBDs of SARS-CoV-1 and SARS-CoV-2 variants, as represented by MAP-1002, are potential COVID-19 vaccine candidates worthy further translational study.

A DNA Vaccine Encoding the Full-Length Spike Protein of Beta Variant (B.1.351) Elicited Broader Cross-Reactive Immune Responses against Other SARS-CoV-2 Variants.

The SARS-CoV-2 pandemic remains an ongoing threat to global health with emerging variants, especially the Omicron variant and its sub-lineages. Although large-scale vaccination worldwide has delivered outstanding achievements for COVID-19 prevention, a declining effectiveness to a different extent in emerging SARS-CoV-2 variants was observed in the vaccinated population. Vaccines eliciting broader spectrum neutralizing antibodies and cellular immune responses are urgently needed and important. To achieve this goal, rational vaccine design, including antigen modeling, screening and combination, vaccine pipelines, and delivery, are keys to developing a next-generation COVID-19 vaccine. In this study, we designed several DNA constructs based on codon-optimized spike coding regions of several SARS-CoV-2 variants and analyzed their cross-reactive antibodies, including neutralizing antibodies, and cellular immune responses against several VOCs in C57BL/6 mice. The results revealed that different SARS-CoV-2 VOCs induced different cross-reactivity; pBeta, a DNA vaccine encoding the spike protein of the Beta variant, elicited broader cross-reactive neutralizing antibodies against other variants including the Omicron variants BA.1 and BA.4/5. This result demonstrates that the spike antigen from the Beta variant potentially serves as one of the antigens for multivalent vaccine design and development against variants of SARS-CoV-2.

Correction: A Truncated Receptor-Binding Domain of MERS-CoV Spike Protein Potently Inhibits MERS-CoV Infection and Induces Strong Neutralizing Antibody Responses: Implication for Developing Therapeutics and Vaccines.

[This corrects the article DOI: 10.1371/journal.pone.0081587.].

An mRNA vaccine encoding Chikungunya virus E2-E1 protein elicits robust neutralizing antibody responses and CTL immune responses.

Arthropod-borne chikungunya virus (CHIKV) infection can cause a debilitating arthritic disease in human. However, there are no specific antiviral drugs and effective licensed vaccines against CHIKV available for clinical use. Here, we developed an mRNA-lipid nanoparticle (mRNA-LNP) vaccine expressing CHIKV E2-E1 antigen, and compared its immunogenicity with soluble recombinant protein sE2-E1 antigen expressed in S2 cells. For comparison, we first showed that recombinant protein antigens mixed with aluminum adjuvant elicit strong antigen-specific humoral immune response and a moderate cellular immune response in C57BL/6 mice. Moreover, sE2-E1 vaccine stimulated 12-23 folds more neutralizing antibodies than sE1 vaccine and sE2 vaccine. Significantly, when E2-E1 gene was delivered by an mRNA-LNP vaccine, not only the better magnitude of neutralizing antibody responses was induced, but also greater cellular immune responses were generated, especially for CD8+ T cell responses. Moreover, E2-E1-LNP induced CD8+ T cells can perform cytotoxic effect in vivo. Considering its better immunogenicity and convenience of preparation, we suggest that more attention should be placed to develop CHIKV E2-E1-LNP mRNA vaccine.

A single nonsynonymous mutation on ZIKV E protein-coding sequences leads to markedly increased neurovirulence in vivo.

Zika virus (ZIKV) can infect a wide range of tissues including the developmental brain of human fetus. Whether specific viral genetic variants are linked to neuropathology is incompletely understood. To address this, we have intracranially serially passaged a clinical ZIKV isolate (SW01) in neonatal mice and discovered variants that exhibit markedly increased virulence and neurotropism. Deep sequencing analysis combining with molecular virology studies revealed that a single 67D (Aspartic acid) to N (Asparagine) substitution on E protein is sufficient to confer the increased virulence and neurotropism in vivo. Notably, virus clones with D67N mutation had higher viral production and caused more severe cytopathic effect (CPE) in human neural astrocytes U251 â€‹cells in vitro, indicating its potential neurological toxicity to human brain. These findings revealed that a single mutation D67N on ZIKV envelope may lead to severe neuro lesion that may help to explain the neurovirulence of ZIKV and suggest monitoring the occurrence of this mutation during nature infection may be important.

A Combined Adjuvant TF-Al Consisting of TFPR1 and Aluminum Hydroxide Augments Strong Humoral and Cellular Immune Responses in Both C57BL/6 and BALB/c Mice.

TFPR1 is a novel adjuvant for protein and peptide antigens, which has been demonstrated in BALB/c mice in our previous studies; however, its adjuvanticity in mice with different genetic backgrounds remains unknown, and its adjuvanticity needs to be improved to fit the requirements for various vaccines. In this study, we first compared the adjuvanticity of TFPR1 in two commonly used inbred mouse strains, BALB/c and C57BL/6 mice, in vitro and in vivo, and demonstrated that TFPR1 activated TLR2 to exert its immune activity in vivo. Next, to prove the feasibility of TFPR1 acting as a major component of combined adjuvants, we prepared a combined adjuvant, TF-Al, by formulating TFPR1 and alum at a certain ratio and compared its adjuvanticity with that of TFPR1 and alum alone using OVA and recombinant HBsAg as model antigens in both BALB/c and C57BL/6 mice. Results showed that TFPR1 acts as an effective vaccine adjuvant in both BALB/c mice and C57BL/6 mice, and further demonstrated the role of TLR2 in the adjuvanticity of TFPR1 in vivo. In addition, we obtained a novel combined adjuvant, TF-Al, based on TFPR1, which can augment antibody and cellular immune responses in mice with different genetic backgrounds, suggesting its promise for vaccine development in the future.

Sensitive and Easy-Read CRISPR Strip for COVID-19 Rapid Point-of-Care Testing.

Rapid and clinically sensitive detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) play an important role in the contact tracing and containment of the COVID-19 pandemic. A recently developed field-deployable clustered regularly interspaced short palindromic repeats (CRISPR) detection assay with lateral flow strips shows promise for point-of-care detection of SARS-CoV-2. However, the limit of detection of paper strip-based assays (10-100 copies/µL) is much lower than that of fluorescence-based detection methods. In this study, we developed an easy-readout and sensitive enhanced (ERASE) strip to visualize the results of CRISPR detection and improve the sensitivity to 1 copy/µL with an unambiguous easy-read result. Using 649 clinical samples from blind specimens collected from patients in China, we validated our ERASE assay for SARS-CoV-2 RNA detection with 90.67% positive predictive agreement and 99.21% negative predictive agreement. In conclusion, our study provided a customized CRISPR strip for use in a simple, rapid, ultrasensitive, and highly specific assay for SARS-CoV-2 detection. (Clinical Trial Registration number: 2020-008-01; [2020]IEC(ZD01); PJ-NBEY-2020-009-01; 2020#34).

CRISPR-Cas13a Cleavage of Dengue Virus NS3 Gene Efficiently Inhibits Viral Replication.

The CRISPR-Cas9 system has been applied to DNA editing with precision in eukaryotic and prokaryotic systems, but it is unable to edit RNA directly. A recently developed CRISPR-Cas13a system has been shown to be capable of effectively knocking down RNA expression in mammalian and plant cells. In this study, we employ the CRISPR-Cas13a system to achieve reprogrammable inactivation of dengue virus in mammalian cells. Quantitative reverse transcription PCR (qRT-PCR), fluorescence-activated cell sorting (FACS), and plaque assays showed that CRISPR RNA (crRNA) targeting the NS3 region led to the greatest viral inhibition among 10 crRNAs targeting different regions along the dengue viral genomic RNA. Deletions and insertions had also been found adjacent to the NS3 region after NS3-crRNA/Cas13a complex transfection. Our results demonstrate that the CRISPR-Cas13a system is a novel and effective technology to inhibit dengue viral replication, suggesting that such a programmable method may be further developed into a novel therapeutic strategy for dengue and other RNA viruses.

TFPR1 acts as an immune regulator and an efficient adjuvant for proteins and peptides by activating immune cells, primarily through TLR2.

Triflin, a non-toxic protein found in the venom of the Habu snake, belongs to the CRISP (cysteine-rich secretory protein) family, which comprises two domains: a C-terminal cysteine-rich domain (CRD) and an N-terminal pathogenesis-related-1 (PR-1) domain. The function of the highly structurally conserved PR-1 domain is unknown. Here, we successfully expressed the PR-1 domain of triflin (hereafter called TFPR1) in E. coli. Animal experiments showed that TFPR1 augmented Th1-biased antibody- and cell-mediated immune responses in mice immunized with two protein antigens (OVA and HBsAg) or a peptide antigen (HIV-1 pep). A flow cytometry-based binding assay and in vitro stimulation with TFPR1 showed that it triggered Th1-biased proinflammatory and immunoregulatory cytokine secretion primarily by binding to B cells and macrophages within the mouse splenocyte population. Quantitative RT-PCR, antibody blocking assays using a specific anti-mTLR2 antibody, and stimulatory experiments in vitro using splenocytes from TLR2-KO mice demonstrated that TFPR1 activated murine immune cells, primarily by stimulating toll-like receptor 2 (TLR2). These results suggest that TFPR1 acts as a novel immune modulator and potent adjuvant primarily by activating TLR2. Thus, the PR-1-based core domain might play a role in immune regulation.

The Integrity of α-ß-α Sandwich Conformation Is Essential for a Novel Adjuvant TFPR1 to Maintain Its Adjuvanticity.

TFPR1 is a novel peptide vaccine adjuvant we recently discovered. To define the structural basis and optimize its application as an adjuvant, we designed three different truncated fragments that have removed dominant B epitopes on TFPR1, and evaluated their capacity to activate bone marrow-derived dendritic cells and their adjuvanticity. Results demonstrated that the integrity of an α-ß-α sandwich conformation is essential for TFPR1 to maintain its immunologic activity and adjuvanticity. We obtained a functional truncated fragment TFPR-ta ranging from 40-168 aa of triflin that has similar adjuvanticity as TFPR1 but with 2-log fold lower immunogenicity. These results demonstrated a novel approach to evaluate and improve the activity of protein-based vaccine adjuvant.

Intranasal immunization with recombinant outer membrane protein A induces protective immune response against Stenotrophomonas maltophilia infection.

Stenotrophomonas maltophilia (S. maltophilia), a multi-drug resistant opportunistic pathogen, is associated with nosocomial and community-acquired infections. Preventive and therapeutic strategies for such infections are greatly needed. In this study, sequence alignment analysis revealed that Outer membrane protein A (OmpA) was highly conserved among S. maltophilia strains but shared no significant similarity with human and mouse proteomes. In mice, intranasal immunization with S. maltophilia recombinant OmpA (rOmpA) without additional adjuvant induced sustained mucosal and systemic rOmpA-specific antibody responses. Treatment with rOmpA stimulated significantly higher levels of secretion of IFN-γ, IL-2, and IL-17A (All P<0.05) from the primary splenocytes isolated from rOmpA-immunized mice than from the primary splenocytes isolated from PBS-immunized mice. Furthermore, mice immunized with rOmpA showed significantly reduced bacterial burden in the lung and reduced levels of pro-inflammatory cytokines (TNF-α and IL-6) in bronchoalveolar lavage fluid (BALF) 24 hours after intranasal S. maltophilia infection, indicating that immunization with rOmpA may have protective effects against S. maltophilia challenge in mice. Our findings suggest that intranasal immunization with rOmpA may induce mucosal and systemic immune responses in mice, trigger Th1- and Th17-mediated cellular immune responses, and thus stimulate host immune defense against S. maltophilia infection. These results also demonstrate that intranasal vaccination may offer an alternative approach to current strategies since it induces a mucosal as well as a systemic immune response.

A novel polyepitope vaccine elicited HIV peptide specific CD4+ T cell responses in HLA-A2/DRB1 transgenic mice.

Human immunodeficiency (HIV) infection is a leading global health problem that causes approximately one million deaths each year. Although antiretroviral therapy can slow down the disease progression and improve the quality of life of infected individuals, it cannot eradicate the virus. A successful vaccine is one of the most cost-effective alternatives to control the incidence and mortality of HIV infection. CD4+ T cells play a key role in orchestrating other forms of human immune responses, therefore, an HIV vaccine that includes a component capable of eliciting CD4+ T cell responses is highly desirable. To this end, we have previously designed a polypeptide vaccine comprised of multiple CD4+ T cell epitopes. In the current study, we tested the immunogenicity of this vaccine in mouse models by using IFN-γELISPOT and intracellular cytokine staining assays. We found that several epitopes in this vaccine elicited CD4+ T cell immune responses in both congenic mice and human HLA-A2/DRB1 transgenic mice. These new epitopes may be further tested for their ability to augment immune responses elicited by other forms of HIV vaccines.

A recombinant multi-epitope protein MEP1 elicits efficient long-term immune responses against HIV-1 infection.

The effective protective HIV vaccine should elicit either protective antibodies or effective T cell response, or both. To improve the efficacy of HIV-1 vaccines, HLA polymorphism and HIV-1 diversity are 2 key factors to be considered for vaccine development. In this study, we expressed a recombinant multi-epitope protein MEP1 which has the same amino acid sequence as a DNA vaccine for Chinese population in our previous report. We found that MEP1 alone could elicit moderate levels of humoral and cellular immune responses, but these responses could not provide protection from challenge with a recombinant virus rTTV-lucgag, which expresses Gag of HIV-1 CRF_07BC. Nevertheless, when MEP1 was immunized with aluminum adjuvant, both humoral and cellular immune responses were significantly increased, and they were protective against virus infection; meanwhile, MEP1 with aluminum not only elicited early (10 d post immunization) but also a long-term (at least 44 weeks post immunization) immune responses in BALB/c mice. These results suggested that MEP1 has the potential to be developed as an effective vaccine candidate, and that suitable adjuvant is necessary for this protein to generate protective immune responses.

Generation of human MHC (HLA-A11/DR1) transgenic mice for vaccine evaluation.

The rapid occurrence of emerging infectious diseases demonstrates an urgent need for a new preclinical experimental model that reliably replicates human immune responses. Here, a new homozygous humanized human leukocyte antigen (HLA)-A11/DR1 transgenic mouse (HLA-A11(+/+)/DR01(+/+)/H-2-ß2m(-/-)/IAß(-/-)) was generated by crossing HLA-A11 transgenic (Tg) mice with HLA-A2(+/+)/DR01(+/+)/H-2-ß2m(-/-)/IAß(-/-) mice. The HLA-A11-restricted immune response of this mouse model was then examined. HLA-A11 Tg mice expressing a chimeric major histocompatibility complex (MHC) molecule comprising the α1, α2, and ß2m domains of human HLA-A11 and the α3 transmembrane and cytoplasmic domains of murine H-2D(b) were generated. The correct integration of HLA-A11 and HLA-DR1 into the genome of the HLA-A11/DR1 Tg mice (which lacked the expression of endogenous H-2-I/II molecules) was then confirmed. Immunizing mice with a recombinant HBV vaccine or a recombinant HIV-1 protein resulted in the generation of IFN-γ-producing cytotoxic T lymphocyte (CTL) and antigen-specific antibodies. The HLA-A11-restricted CTL response was directed at HLA immunodominant epitopes. These mice represent a versatile animal model for studying the immunogenicity of HLA CTL epitopes in the absence of a murine MHC response. The established animal model will also be useful for evaluating and optimizing T cell-based vaccines and for studying differences in antigen processing between mice and humans.

Multi-Organ Damage in Human Dipeptidyl Peptidase 4 Transgenic Mice Infected with Middle East Respiratory Syndrome-Coronavirus.

The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) causes severe acute respiratory failure and considerable extrapumonary organ dysfuction with substantial high mortality. For the limited number of autopsy reports, small animal models are urgently needed to study the mechanisms of MERS-CoV infection and pathogenesis of the disease and to evaluate the efficacy of therapeutics against MERS-CoV infection. In this study, we developed a transgenic mouse model globally expressing codon-optimized human dipeptidyl peptidase 4 (hDPP4), the receptor for MERS-CoV. After intranasal inoculation with MERS-CoV, the mice rapidly developed severe pneumonia and multi-organ damage, with viral replication being detected in the lungs on day 5 and in the lungs, kidneys and brains on day 9 post-infection. In addition, the mice exhibited systemic inflammation with mild to severe pneumonia accompanied by the injury of liver, kidney and spleen with neutrophil and macrophage infiltration. Importantly, the mice exhibited symptoms of paralysis with high viral burden and viral positive neurons on day 9. Taken together, this study characterizes the tropism of MERS-CoV upon infection. Importantly, this hDPP4-expressing transgenic mouse model will be applicable for studying the pathogenesis of MERS-CoV infection and investigating the efficacy of vaccines and antiviral agents designed to combat MERS-CoV infection.

Up-regulation of human cervical cancer proto-oncogene contributes to hepatitis B virus-induced malignant transformation of hepatocyte by down-regulating E-cadherin.

Hepatocellular carcinoma (HCC) is one of the most fatal human malignancies, Human cervical cancer proto-oncogene (HCCR) aberrantly expressed in a number of malignant tumors, including HCC. HCC is associated with Hepatitis B virus (HBV) infection in a large percentage of cases. To explore the regulation and function of HCCR expression in the development of HCC, we detected HCCR expression in HBV expressing hepatocytes. Results showed that the expression of HCCR was higher in HBV-expressing hepatocytes than that in control cells. Examining different components of HBV revealed that the HBx promotes HCCR expression in hepatocytes via the T-cell factor (TCF)/ß-catenin pathway. HCCR expression in HBx transgenic mice increased with as the mice aged and developed tumors. We also found that overexpression of HCCR in hepatocytes promoted cell proliferation, migration, and invasion and reduced cell adhesion. Suppressing HCCR expression abolished the effect of HBx-induced hepatocyte growth. In addition, HCCR represses the expression of E-cadherin by inhibition its promoter activity, which might correlate with the effects of HCCR in hepatocytes. Taken together, these results demonstrate that HBx-HCCR-E-cadherin regulation pathway might play an important role in HBV-induced hepatocarcinogenesis. They also imply that HCCR is a potential risk marker for HCC and/or a potential therapeutic target.

In silico design of a DNA-based HIV-1 multi-epitope vaccine for Chinese populations.

The development of an HIV-1 vaccine that is capable of inducing effective and broadly cross-reactive humoral and cellular immune responses remains a challenging task because of the extensive diversity of HIV-1, the difference of virus subtypes (clades) in different geographical regions, and the polymorphism of human leukocyte antigens (HLA). We performed an in silico design of 3 DNA vaccines, designated pJW4303-MEG1, pJW4303-MEG2 and pJW4303-MEG3, encoding multi-epitopes that are highly conserved within the HIV-1 subtypes most prevalent in China and can be recognized through HLA alleles dominant in China. The pJW4303-MEG1-encoded protein consisted of one Th epitope in Env, and one, 2, and 6 epitopes in Pol, Env, and Gag proteins, respectively, with a GGGS linker sequence between epitopes. The pJW4303-MEG2-encoded protein contained similar epitopes in a different order, but with the same linker as pJW4303-MEG1. The pJW4303-MEG3-encoded protein contained the same epitopes in the same order as that of pJW4303-MEG2, but with a different linker sequence (AAY). To evaluate immunogenicity, mice were immunized intramuscularly with these DNA vaccines. Both pJW4303-MEG1 and pJW4303-MEG2 vaccines induced equally potent humoral and cellular immune responses in the vaccinated mice, while pJW4303-MEG3 did not induce immune responses. These results indicate that both epitope and linker sequences are important in designing effective epitope-based vaccines against HIV-1 and other viruses.

Upregulation of microRNA-146a by hepatitis B virus X protein contributes to hepatitis development by downregulating complement factor H.

UNLABELLED: Hepatic injuries in hepatitis B virus (HBV) patients are caused by immune responses of the host. In our previous study, microRNA-146a (miR-146a), an innate immunity-related miRNA, and complement factor H (CFH), an important negative regulator of the alternative pathway of complement activation, were differentially expressed in HBV-expressing and HBV-free hepatocytes. Here, the roles of these factors in HBV-related liver inflammation were analyzed in detail. The expression levels of miR-146a and CFH in HBV-expressing hepatocytes were assessed via analyses of hepatocyte cell lines, transgenic mice, adenovirus-infected mice, and HBV-positive human liver samples. The expression level of miR-146a was upregulated in HBV-expressing Huh-7 hepatocytes, HBV-expressing mice, and patients with HBV infection. Further results demonstrated that the HBV X protein (HBx) was responsible for its effects on miR-146a expression through NF-κB-mediated enhancement of miR-146a promoter activity. HBV/HBx also downregulated the expression of CFH mRNA in hepatocyte cell lines and the livers of humans and transgenic mice. Furthermore, overexpression and inhibition of miR-146a in Huh-7 cells downregulated and upregulated CFH mRNA levels, respectively. Luciferase reporter assays demonstrated that miR-146a downregulated CFH mRNA expression in hepatocytes via 3'-untranslated-region (UTR) pairing. The overall effect of this process in vivo is to promote liver inflammation. These results demonstrate that the HBx-miR-146a-CFH-complement activation regulation pathway might play an important role in the immunopathogenesis of chronic HBV infection. These findings have important implications for understanding the immunopathogenesis of chronic hepatitis B and developing effective therapeutic interventions. IMPORTANCE: Hepatitis B virus (HBV) remains an important pathogen and can cause severe liver diseases, including hepatitis, liver cirrhosis, and hepatocellular carcinoma. Although HBV was found in 1966, the molecular mechanisms of pathogenesis are still poorly understood. In the present study, we found that the HBV X protein (HBx) promoted the expression of miR-146a, an innate immunity-related miRNA, through the NF-κB signal pathway and that increasingly expressed miR-146a downregulated its target complement factor H (CFH), an important negative regulator of the complement alternative pathway, leading to the promotion of liver inflammation. We demonstrated that the HBx-miR-146a-CFH-complement activation regulation pathway is potentially an important mechanism of immunopathogenesis caused by chronic HBV infection. Our data provide a novel molecular mechanism of HBV pathogenesis and thus help to understand the correlations between the complement system, an important part of innate immunity, and HBV-associated disease. These findings will also be important to identify potential therapeutic targets for HBV infection.

A truncated fragment of Ov-ASP-1 consisting of the core pathogenesis-related-1 (PR-1) domain maintains adjuvanticity as the full-length protein.

The Onchocerca volvulus activation-associated secreted protein-1 (Ov-ASP-1) has good adjuvanticity for a variety of antigens and vaccines, probably due to its ability activate antigen-processing cells (APCs). However, the functional domain of Ov-ASP-1 as an adjuvant is not clearly defined. Based on the structural prediction of this protein family, we constructed a 16-kDa recombinant protein of Ov-ASP-1 that contains only the core pathogenesis-related-1 (PR-1) domain (residues 10-153), designated ASPPR. We found that ASPPR exhibits adjuvanticity similar to that of the full-length Ov-ASP-1 (residues 10-220) for various antigens, including ovalbumin (OVA), HBsAg protein antigen, and the HIV peptide 5 (Pep5) antigen, but it is more suitable for vaccine design in ASPPR-antigen fusion proteins, and more stable in PBS than Ov-ASP-1 stored at -70 °C. These results suggest that ASPPR might be the functional region of Ov-ASP-1 as an adjuvant, and therefore could be developed as an adjuvant for human use.