Augmentation of alphavirus vector-induced human papilloma virus-specific immune and anti-tumour responses by co-expression of interleukin-12

To enhance the efficacy of a therapeutic immunisation strategy against human papillomavirus-induced cervical cancer we evaluated the adjuvant effect of interleukin-12 (IL12) expressed by a Semliki Forest virus vector (SFV) in mice. Depending on the dose and schedule, SFV-IL12 stimulated antigen-specific CTL responses elicited upon immunisation with recombinant SFV expressing HPV16-E6E7 (SFVeE6,7). SFVeE6,7–CTL and anti-tumour activity were enhanced by a low dose of SFV-IL12 to the prime immunisation. Using higher dosages these activities were reduced. Addition of SFV-IL12 to the booster immunisation further reduced the efficacy of the SFVeE6,7 immunisation. In transgenic mice, tolerant for HPV16-E6E7, SFV-IL12 also stimulated SFVeE6,7-induced CTL responses. In conclusion, SFV-IL12 can enhance antigen-specific immune responses. Yet, prudence is called for when considering co-administration of SFV-IL12 to a vaccine, as the enhancement of cell-mediated immune responses greatly depends on dosage and schedule.     

海南岛两株甲病毒基因组3＇末端核苷酸序列的克隆与分析

目的 从分子水平鉴定海南省分离的两株甲病毒（ＨＢｂ１７和Ｍ１病毒）。方法 采用ＲＴ－ＰＣＲ方法,分别扩增两株病毒基因组的３’末端核苷酸序列,扩增产物经亚克隆,筛选重组子并测定核苷酸序列对序列进行分析。结果 ＨＢｂ１７和Ｍ１病毒分别扩增出约１．６ｋｂ和１．３ｋｂ的特异片段。序列分析表明,在３’末端非翻译区ＨＢｂ１７病毒和Ｍ１病毒与罗斯病毒（国际标准株Ｔ４８病毒）核苷酸同源性为９９％,Ｍ１病毒与鹭山病     

Genomic analysis of a Chinese isolate of Getah-like virus and its phylogenetic relationship with other Alphaviruses

An alphavirus, M-1 strain, was isolated from a pool of culicine mosquitoes collected in Hainan island of China during an arbovirus survey in 1964. In the present study, we determined the complete nucleotide sequence of the M-1 strain using RT-PCR and RACE techniques. The M-1 genome is 11,690 nucleotides (nt) in length and contains two open reading frames (ORFs) encoding four nonstructural proteins and five structural proteins, respectively. Searches using Blast and comparison analyses suggested that M-1 is closely linked to Sagiyama virus (SAGV, AB032553) with 98% identity and Getah viruse (GETV, AY702913) with 97.8% identity in the full-length nucleotide sequence. However, compared with SAGV, there is 1 deletion (3 nucleotides in length) in the Capsid region, a deletion in the 3′ untranslated region (10 nucleotides in length) and 2 insertions in the 3′ untranslated region involving a total of 5 nucleotides. Interestingly, from the 5′ UTR to the end of coding region, M-1 share the highest identity with GETV, even though the identity of 3′ UTR drops dramatically to 76.2%. Furthermore, phylogenetic analysis based on the complete genomic sequences and sequences for structural or non-structural proteins of M-1 and 15 alphaviruses showed that M-1 grouped with GETV first and then grouped together with SAGV. Based on the comparison analysis and phylogenetic analysis, we conclude that M-1 strain can be considered as a strain that is a Chinese isolate of Getah-like virus. Jin-Sheng Wen and Wen-Zhong Zhao equally contributed to this work. The nucleotide sequence data reported in this article have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number: EF011023.     

Characteristics of alpha/beta interferon induction after infection of murine fibroblasts with wild-type and mutant alphaviruses

We examined the characteristics of interferon alpha/beta (IFN-α/β) induction after alphavirus or control Sendai virus (SeV) infection of murine fibroblasts (MEFs). As expected, SeV infection of wild-type (wt) MEFs resulted in strong dimerization of IRF3 and the production of high levels of IFN-α/β. In contrast, infection of MEFs with multiple alphaviruses failed to elicit detectable IFN-α/β. In more detailed studies, Sindbis virus (SINV) infection caused dimerization and nuclear migration of IRF3, but minimal IFN-β promoter activity, although surprisingly, the infected cells were competent for IFN production by other stimuli early after infection. A SINV mutant defective in host macromolecular synthesis shutoff induced IFN-α/β in the MEF cultures dependent upon the activities of the TBK1 IRF3 activating kinase and host pattern recognition receptors (PRRs) PKR and MDA5 but not RIG-I. These results suggest that wild-type alphaviruses antagonize IFN induction after IRF3 activation but also may avoid detection by host PRRs early after infection.     

Alphavirus production is inhibited in neurofibromin 1-deficient cells through activated RAS signalling

Virus-host interactions essential for alphavirus pathogenesis are poorly understood. To address this shortcoming, we coupled retrovirus insertional mutagenesis and a cell survival selection strategy to generate clonal cell lines broadly resistant to Sindbis virus (SINV) and other alphaviruses. Resistant cells had significantly impaired SINV production relative to wild-type (WT) cells, although virus binding and fusion events were similar in both sets of cells. Analysis of the retroviral integration sites identified the neurofibromin 1 (NF1) gene as disrupted in alphavirus-resistant cell lines. Subsequent analysis indicated that expression of NF1 was significantly reduced in alphavirus-resistant cells. Importantly, independent down-regulation of NF1 expression in WT HEK 293 cells decreased virus production and increased cell viability during SINV infection, relative to infected WT cells. Additionally, we observed hyperactive RAS signalling in the resistant HEK 293 cells, which was anticipated because NF1 is a negative regulator of RAS. Expression of constitutively active RAS (HRAS-G12V) in a WT HEK 293 cell line resulted in a marked delay in virus production, compared with infected cells transfected with parental plasmid or dominant-negative RAS (HRAS-S17N). This work highlights novel host cell determinants required for alphavirus pathogenesis and suggests that RAS signalling may play an important role in neuronal susceptibility to SINV infection.     

蚊类携带甲病毒属病毒CODEHOP RT-PCR检测方珐的建立

目的建立蚊类感染甲病毒属病毒的CODEHOPRT—PCR检测方法。方法依据甲病毒属病毒非结构蛋白氨基酸序列,设计1对CODEHOP引物,建立甲病毒属病毒一步RT—PCR检测方法,分析该引物在对不同种甲病毒基因序列上的结合位点及所能获得的产物序列大小;通过检测甲病毒属基孔肯亚病毒JC2012株,评价该方法的特异性和灵敏度：对JC2012扩增产物进行Blast同源性比对。结果建立的CODEHOPRT-PCR方法可特异性扩增基孔肯亚病毒核酸,目的片段的大小与预期结果相符,核酸的最小检出量为56．7pg。经Blast比对,结果与Genebank公布的CHIKVJC2012序列结果一致。同时从GenBank获得的22种甲病毒均存在CODEHOP引物结合位点．扩增产物大小在510～550bp之间。结论建立的甲病毒CODEHOPRT—PCR检测方法敏感、特异,可用于蚊类甲病毒感染检测。     

Infected dendritic cells are sufficient to mediate the adjuvant activity generated by Venezuelan equine encephalitis virus replicon particles

Replicon particles derived from Venezuelan equine encephalitis virus (VEE) are infectious non-propagating particles which act as a safe and potent systemic, mucosal, and cellular adjuvant when delivered with antigen. VEE and VEE replicon particles (VRP) can target multiple cell types including dendritic cells (DCs). The role of these cell types in VRP adjuvant activity has not been previously evaluated, and for these studies we focused on the contribution of DCs to the response to VRP. By analysis of VRP targeting in the draining lymph node, we found that VRP induced rapid recruitment of TNF-secreting monocyte-derived inflammatory dendritic cells. VRP preferentially infected these inflammatory DCs as well as classical DCs and macrophages, with less efficient infection of other cell types. DC depletion suggested that the interaction of VRP with classical DCs was required for recruitment of inflammatory DCs, induction of high levels of many cytokines, and for stable transport of VRP to the draining lymph node. Additionally, in vitro-infected DCs enhanced antigen-specific responses by CD4 and CD8 T cells. By transfer of VRP-infected DCs into mice we showed that these DCs generated an inflammatory state in the draining lymph node similar to that achieved by VRP injection. Most importantly, VRP-infected DCs were sufficient to establish robust adjuvant activity in mice comparable to that produced by VRP injection. These findings indicate that VRP infect, recruit and activate both classical and inflammatory DCs, and those DCs become mediators of the VRP adjuvant activity.     

Semliki Forest virus-based immunotherapy for cancer

ABSTRACT

Introduction: Immunotherapy has been introduced as a modern alternative for the treatment of various cancers, including the stimulation of the immune system by introduction of immunostimulatory molecules. Application of viral and non-viral vectors have provided a substantial contribution to improved delivery and expression of these immunostimulators.

Areas covered: Alphavirus vectors, based on Semliki Forest virus, have allowed immunization with self-replicating RNA, recombinant virus particles, and layered DNA/RNA vectors. The attractive features of alphaviruses comprise their broad host range and extreme RNA replication in infected cells resulting in very high recombinant protein expression levels providing enhanced immune responses and an excellent basis for immunotherapy.

Expert opinion: Immunization studies in animal tumor models have elicited strong humoral and cellular immune response, have provided prophylactic protection against tumor challenges, and have generated therapeutic efficacy in tumor-bearing animals. Clinical trials have indicated safe use of alphavirus vectors, making them attractive for cancer immunotherapy.     

云南省澜沧江下游地区人及动物血清虫媒病毒抗体调查

目的 :对澜沧江下游地区的思茅和西双版纳两地州进行虫媒病毒抗体调查 ,了解当地虫媒病毒的流行情况。方法 :采集人及动物血清 ,用ELISA和血凝抑制试验检测抗体。结果 :发热病人血清中 ,乙型脑炎 (JE)、登革 3型 (DEN3)、DEN4、基孔肯雅 (CHIK)、辛德毕斯 (SIN)、贝巴鲁 (BEB)、雪鞋野兔 (SSH)和巴泰 (BAT)病毒抗体的阳性率依次为 7.5 0 %、5 .0 0 %、1.6 7%、3.33%、2 .5 0 %、1.6 7%、5 .0 0 %和 4 .17% ;未查到DEN1、DEN2和寨卡 (Zika)病毒抗体。正常人血清JE、墨累山谷脑炎 (MVE)、库京 (KUN)、DEN、圣路易脑炎 (SLE)、科萨努尔森林病 (KFD)、森林脑炎 (RSSE)、兰加特 (LAG)、波瓦生 (POW )、马雅罗 (MAY)、CHIK、SIN、委内瑞拉马脑炎 (VEE)、西门利克森林病(SFD)和格塔 (GET)病毒抗体阳性率依次为 34.88%、、35 .0 3%、2 0 .81%、9.2 5 %、1.78%、12 .4 6 %、4 .76 %、2 .85 %、2 .4 9%、2 1.71%、11.0 3%、1.93%、2 .4 9%、2 .4 9%、3.2 0 %。从恒河猴血清中检出DEN、KUN、RSSE、KFD、POW、LAG、CHIK和GET病毒抗体 ,阳性率以DEN4、KUN和RSSE为高。棕果蝠血清中查到JE、DEN4、RSSE、POW和LAG病毒抗体 ,JE的阳性率最高。猪血清中查到JE、DEN和CHIK抗体。黄胸鼠血清检出JE、KFD、CHIK和SIN病毒抗体 ,