Caijun Sun

MicroRNAs reduce tumor growth and contribute to enhance cytotoxicity induced by gefitinib in non-small cell lung cancer.

MicroRNAs (miRNAs) have emerged as key post-transcriptional regulators of gene expression, involved in diverse physiological and pathological processes. An oncogenic or tumor-suppressive miRNA may have potential as a therapeutic target to control cancers. Gefitinib is a tyrosine kinase inhibitor that targets epidermal growth factor receptor (EGFR). H460 and A549 cells with EGFR receptor-independent over-activation of protein kinase B (Akt) or extracellular signal-regulated kinases (ERK) are significantly resistant to gefitinib. The first aim of this study was to confirm a role for three miRNAs (let-7a, hsa-miR-126, and hsa-miR-145) in the inhibition of proliferation in non-small cell lung cancer (NSCLC) cells. A second aim was to evaluate three miRNAs for their abilities to overcome cellular resistance and enhance the gefitinib cytotoxicity. The expression of miRNAs was estimated by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell proliferation was examined by sulforhodamine B assay and tumor xenografts were measured in SCID/beige mice. The activation of Akt and ERK was observed by Western blotting. Forced expression of individual miRNA suppressed the growth of two cell lines and xenografts. The effect varied among different miRNAs and cells. Restoration of hsa-miR-126 more obviously inhibited cell growth than did restoration of hsa-miR-145 in both cells, and the suppressive effect was more significant in H460 xenografts than in A549 xenografts. Western blotting revealed that the inhibition of cell proliferation resulted from the inhibition of the activation of Akt and ERK. Moreover, forced expression of miRNAs contributed to enhanced cytotoxicity induced by gefitinib in lung cancer cells; especially in hsa-miR-126, the highest value of half max inhibitory (IC50) was increased sixfold. These findings confirm that tumor-suppressive miRNAs can inhibit the growth of NSCLC cells and enhance the targeted agents cytotoxicity, suggesting novel potential approaches to an improvement in chemotherapy.

Visualizing influenza virus infection in living mice

Preventing and treating influenza virus infection remain a challenge because of incomplete understanding of the host–pathogen interactions, limited therapeutics and lack of a universal vaccine. So far, methods for monitoring the course of infection with influenza virus in real time in living animals are lacking. Here we report the visualization of influenza viral infection in living mice using an engineered replication-competent influenza A virus carrying luciferase reporter gene. After intranasal inoculation, bioluminescence can be detected in the chest and nasopharyngeal passage of living mice. The intensity of bioluminescence in the chest correlates with the dosage of infection and the viral load in the lung. Bioluminescence in the chest of infected mice diminishes on antiviral treatment. This work provides a novel approach that enables real-time study of influenza virus infection and effects of antiviral therapeutics in living animals.

Epidemiology of adenovirus type 5 neutralizing antibodies in healthy people and AIDS patients in Guangzhou, southern China

Recombinant adenovirus serotype 5 (Ad5) viruses have been extensively explored as vectors for vaccination or gene therapy. However, one major obstacle to their clinical application is the high prevalence of preexisting anti-Ad5 immunity resulting from natural infection. It has been reported that there are geographic variations in the prevalence of natural adenovirus infection. In the present study, we investigated the seroprevalence of Ad5 in Guangzhou, southern China by measuring the Ad5 neutralizing antibodies in blood samples collected from several sites. The seroprevalence was 77.34% in the general healthy population. The seroprevalence and antibody titers increased with age, with the older population (41-72 years old) having the highest seropositivity (84.8%) and percentage (54.4%) of high Ad5 neutralizing antibody titers (>1000). The dynamics of Ad5 neutralizing antibodies were stable and persistent over the course of eight months. Furthermore, the seroprevalence of Ad5 in the HIV-infected AIDS patients was investigated and there was no significant difference from the general healthy population. Our survey provides useful insights for the future development of Ad5-based vaccination and gene therapy.

Generation of Replication-Competent Recombinant Influenza A Viruses Carrying a Reporter Gene Harbored in the Neuraminidase Segment

ABSTRACT Replication-competent influenza viruses carrying reporter genes are of great use for basic research, screening of antiviral drugs, and neutralizing of antibodies. In this study, two recombinant influenza A viruses with a neuraminidase (NA) segment harboring enhanced green fluorescent protein (EGFP) in the background of A/PR/8/34 (PR8) were generated. The viral RNA (vRNA)-specific packaging signals for NA were largely retained for efficient packaging. An “autocleave” 2A peptide sequence, which was inserted at the N terminus or the COOH terminus of NA to link with EGFP, enabled NA and EGFP to be expressed monocistronically. Further analysis demonstrated that both viruses, named rPR8-EGFP+NA and rPR8-NA+EGPF, although with some characteristic differences in growth and EGFP expression, could replicate in noncomplementary cells and propagate to large quantities while maintaining genome stability after multiple passages in embryonated eggs. These replication-competent influenza viruses carrying reporter genes are a great addition to the tool set for developing antiviral therapeutics and vaccines and for in vivo studies of viral dissemination and pathogenicity.

Mucosal Priming with a Replicating-Vaccinia Virus-Based Vaccine Elicits Protective Immunity to Simian Immunodeficiency Virus Challenge in Rhesus Monkeys

ABSTRACT Mucosal surfaces are not targeted by most human immunodeficiency virus type 1 (HIV-1) vaccines, despite being major routes for HIV-1 transmission. Here we report a novel vaccination regimen consisting of a mucosal prime with a modified replicating vaccinia virus Tiantan strain (MVTTSIVgpe) and an intramuscular boost with a nonreplicating adenovirus strain (Ad5SIVgpe). This regimen elicited robust cellular immune responses with enhanced magnitudes, sustainability, and polyfunctionality, as well as higher titers of neutralizing antibodies against the simian immunodeficiency virus SIVmac1A11 in rhesus monkeys. The reductions in peak and set-point viral loads were significant in most animals, with one other animal being protected fully from high-dose intrarectal inoculation of SIVmac239. Furthermore, the animals vaccinated with this regimen were healthy, while ∼75% of control animals developed simian AIDS. The protective effects correlated with the vaccine-elicited SIV-specific CD8+ T cell responses against Gag and Pol. Our study provides a novel strategy for developing an HIV-1 vaccine by using the combination of a replicating vector and mucosal priming.

Induction of balance and breadth in the immune response is beneficial for the control of SIVmac239 replication in rhesus monkeys.

OBJECTIVES The aim of this study was to induce cellular and humoral responses with enhanced breadth and more balanced magnitude as a possible approach for an effective HIV vaccine. METHODS All nine of the SIVmac239 genes (gag, pol, env, nef, vif, vpx, vpr, rev and tat) were optimized for mammalian expression, synthesized and cloned into recombinant adenovirus type 5 (Ad5). These vectors were used as a vaccine regimen, and the immunogenicity and immune protection of this regimen was assessed in murine and macaques. RESULTS A vaccine regimen including all nine genes of the SIVmac239 virus was developed, and it was demonstrated that in contrast to single antigen vaccination, the total SIV antigen regimen more effectively elicited the balanced and broad immune responses in murine and macaques. Moreover, the responses afforded effective immune control against infection and replication of the highly pathogenic SIVmac239. CONCLUSIONS Induction of balance and breadth in the immune response is beneficial in controlling SIVmac239 replication in rhesus monkeys. This study provides insight for the future development of an effective HIV vaccine.

Regulation of SIV antigen-specific CD4+ T cellular immunity via autophagosome-mediated MHC II molecule-targeting antigen presentation in mice.

CD4+ T cell-mediated immunity has increasingly received attention due to its contribution in the control of HIV viral replication; therefore, it is of great significance to improve CD4+ T cell responses to enhance the efficacy of HIV vaccines. Recent studies have suggested that macroautophagy plays a crucial role in modulating adaptive immune responses toward CD4+ T cells or CD8+ T cells. In the present study, a new strategy based on a macroautophagy degradation mechanism is investigated to enhance CD4+ T cell responses against the HIV/SIV gag antigen. Our results showed that when fused to the autophagosome-associated LC3b protein, SIVgag protein can be functionally targeted to autophagosomes, processed by autophagy-mediated degradation in autolysosomes/lysosomes, presented to MHC II compartments and elicit effective potential CD4 T cell responses in vitro. Importantly, compared with the SIVgag protein alone, SIVgag-LC3b fusion antigen can induce a stronger antigen-specific CD4+ T cell response in mice, which is characterized by an enhanced magnitude and polyfunctionality. This study provides insight for the immunological modulation between viral and mammalian cells via autophagy, and it also presents an alternative strategy for the design of new antigens in the development of effective HIV vaccines.

Proton channel activity of influenza A virus matrix protein 2 contributes to autophagy arrest

ABSTRACT Influenza A virus infection can arrest autophagy, as evidenced by autophagosome accumulation in infected cells. Here, we report that this autophagosome accumulation can be inhibited by amantadine, an antiviral proton channel inhibitor, in amantadine-sensitive virus infected cells or cells expressing influenza A virus matrix protein 2 (M2). Thus, M2 proton channel activity plays a role in blocking the fusion of autophagosomes with lysosomes, which might be a key mechanism for arresting autophagy.

Vaccine Engineering with Dual-Functional Mineral Shell: A Promising Strategy to Overcome Preexisting Immunity.

Dual-functional biomineral-vaccine core-shell nanohybrids are obtained using recombinant adenovirus serotype 5 (rAd5) as templates, which efficiently masks the neutralizing epitope of vaccines and preserve their original immunogenicity. The versatile vaccine hybrid can evade the preexisting anti-Ad5 immunity, leading to boosted multifunctional antigen-specific cytokine-secreting T cell responses and presenting promising applications of vaccine-material hybrid for the rational design of vaccines.

Rapid Generation of Human-Like Neutralizing Monoclonal Antibodies in Urgent Preparedness for Influenza Pandemics and Virulent Infectious Diseases

Background The outbreaks of emerging infectious diseases caused by pathogens such as SARS coronavirus, H5N1, H1N1, and recently H7N9 influenza viruses, have been associated with significant mortality and morbidity in humans. Neutralizing antibodies from individuals who have recovered from an infection confer therapeutic protection to others infected with the same pathogen. However, survivors may not always be available for providing plasma or for the cloning of monoclonal antibodies (mAbs). Methodology/Principal Findings The genome and the immunoglobulin genes in rhesus macaques and humans are highly homologous; therefore, we investigated whether neutralizing mAbs that are highly homologous to those of humans (human-like) could be generated. Using the H5N1 influenza virus as a model, we first immunized rhesus macaques with recombinant adenoviruses carrying a synthetic gene encoding hemagglutinin (HA). Following screening an antibody phage display library derived from the B cells of immunized monkeys, we cloned selected macaque immunoglobulin heavy chain and light chain variable regions into the human IgG constant region, which generated human-macaque chimeric mAbs exhibiting over 97% homology to human antibodies. Selected mAbs demonstrated potent neutralizing activities against three clades (0, 1, 2) of the H5N1 influenza viruses. The in vivo protection experiments demonstrated that the mAbs effectively protected the mice even when administered up to 3 days after infection with H5N1 influenza virus. In particular, mAb 4E6 demonstrated sub-picomolar binding affinity to HA and superior in vivo protection efficacy without the loss of body weight and obvious lung damage. The analysis of the 4E6 escape mutants demonstrated that the 4E6 antibody bound to a conserved epitope region containing two amino acids on the globular head of HA. Conclusions/Significance Our study demonstrated the generation of neutralizing mAbs for potential application in humans in urgent preparedness against outbreaks of new influenza infections or other virulent infectious diseases.