Efficacy of recombinant Marek’s disease virus vectored vaccines with computationally optimized broadly reactive antigen (COBRA) hemagglutinin insert against genetically diverse H5 high pathogenicity avian influenza viruses

The genetic and antigenic drift associated with the high pathogenicity avian influenza (HPAI) viruses of Goose/Guangdong (Gs/GD) lineage and the emergence of vaccine-resistant field viruses underscores the need for a broadly protective H5 influenza A vaccine. Here, we tested experimental vector herpesvirus of turkey (vHVT)-H5 vaccines containing either wild-type clade 2.3.4.4A-derived H5 inserts or computationally optimized broadly reactive antigen (COBRA) inserts with challenge by homologous and genetically divergent H5 HPAI Gs/GD lineage viruses in chickens. Direct assessment of protection was confirmed for all the tested constructs, which provided clinical protection against the homologous and heterologous H5 HPAI Gs/GD challenge viruses and significantly decreased oropharyngeal shedding titers compared to the sham vaccine. The cross reactivity was assessed by hemagglutinin inhibition (HI) and focus reduction assay against a panel of phylogenetically and antigenically diverse H5 strains. The COBRA-derived H5 inserts elicited antibody responses against antigenically diverse strains, while the wild-type-derived H5 vaccines elicited protection mostly against close antigenically related clades 2.3.4.4A and 2.3.4.4D viruses. In conclusion, the HVT vector, a widely used replicating vaccine platform in poultry, with H5 insert provides clinical protection and significant reduction of viral shedding against homologous and heterologous challenge. In addition, the COBRA-derived inserts have the potential to be used against antigenically distinct co-circulating viruses and future drift variants.     

Stabilization and formulation of a recombinant Human Cytomegalovirus vector for use as a candidate HIV-1 vaccine

Live attenuated viral vaccine/vector candidates are inherently unstable and infectivity titer losses can readily occur without defining appropriate formulations, storage conditions and clinical handling practices. During initial process development of a candidate vaccine against HIV-1 using a recombinant Human Cytomegalovirus vector (rHCMV-1), large vector titer losses were observed after storage at 4 °C and after undergoing freeze-thaw. Thus, the goal of this work was to develop candidate frozen liquid formulations of rHCMV-1 with improved freeze-thaw and short-term liquid stability for potential use in early clinical trials. To this end, a virus stability screening protocol was developed including use of a rapid, in vitro cell-based immunofluorescence focus assay to quantitate viral titers. A library of ∼50 pharmaceutical excipients (from various known classes of additives) were evaluated for their effect on vector stability after freeze-thaw cycling or incubation at 4 °C for several days. Certain additives including sugars and polymers (e.g., trehalose, sucrose, sorbitol, hydrolyzed gelatin, dextran 40) as well as removal of NaCl (lower ionic strength) protected rHCMV-1 against freeze-thaw mediated losses in viral titers. Optimized solution conditions (e.g., solution pH, buffers and sugar type) slowed the rate of rHCMV-1 titer losses in the liquid state at 4 °C. After evaluating various excipient combinations, three new candidate formulations were designed and rHCMV-1 stability was benchmarked against both the currently-used and a previously reported formulation. The new candidate formulations were significantly more stable in terms of reducing rHCMV-1 titer losses after 5 freeze-thaw cycles or incubation at 4 °C for 30 days. This case study highlights the utility of semi-empirical design of frozen liquid formulations of a live viral vaccine candidate, where protection against infectivity titer losses due to freeze-thaw and short-term liquid storage are sufficient to enable more rapid initiation of early clinical trials.     

CD8+ T cells induced by adenovirus-vectored vaccine are capable of preventing establishment of latent murine γ-herpesvirus 68 infection

CD8+ T cells are known to control infections, but their role in preventing latent infection from establishing has not been thoroughly investigated.We hypothesized that a potent CD8+ T cell response patrolling the mucosal viral entry points could kill the first infected cells and thereby abrogate the infection before latency is established.To investigate this, replication deficient adenovirus serotype 5 vectors encoding murine γ-herpesvirus-68 CD8+ T cell epitopes linked to the T cell adjuvant Invariant chain, were developed. We show that intranasal vaccination of mice reduces the risk of establishment of latent infection from multiple intranasal ID50 challenges with murine γ-herpesvirus-68 by 81% per exposure at 14 days post vaccination. Protection waned over time, but immune responses were extended by heterologous prime-boost vaccination applied simultaneously intramuscularly and intranasally, and animals vaccinated 66 days prior to challenge showed a strong trend of long-term protection.Our data provides evidence that CD8+ T cells are able to protect against establishment of latent infection. Although the protective efficacy is difficult to maintain over time, this proof-of-concept study suggests a role for a CD8+ T cell arm in future vaccine strategies against latent human viral infections caused by pathogens such as HIV and multiple herpes virus.     

Protective effects of pharmacological therapies in animal models of multiple sclerosis: a review of studies 2014–2019

Multiple sclerosis(MS)is an inflammatory demyelinating disease of the central nervous system.The disability caused by inflammatory demyelination clinically dominates the early stages of relapsing-remitting MS and is reversible.Once there is considerable loss of axons,MS patients enter a secondary progressive stage.Disease-modifying drugs currently in use for MS suppress the immune system and reduce relapse rates but are not effective in the progressive stage.Various animal models of MS(mostly mouse and rat)have been established and proved useful in studying the disease process and response to therapy.The experimental autoimmune encephalomyelitis animal studies reviewed here showed that a chronic progressive disease can be induced by immunization with appropriate amounts of myelin oligodendrocyte glycoprotein together with mycobacterium tuberculosis and pertussis toxin in Freund's adjuvant.The clinical manifestations of autoimmune encephalomyelitis disease were prevented or reduced by treatment with certain pharmacological agents given prior to,at,or after peak disease,and the agents had protective effects as shown by inhibiting demyelination and damage to neurons,axons and oligodendrocytes.In the cuprizone-induced toxicity animal studies,the pharmacological agents tested were able to promote remyelination and increase the number of oligodendrocytes when administered therapeutically or prophylactically.A monoclonal IgM antibody protected axons in the spinal cord and preserved motor function in animals inoculated with Theiler's murine encephalomyelitis virus.In all these studies the pharmacological agents were administered singly.A combination therapy may be more effective,especially using agents that target neuroinflammation and neurodegeneration,as they may exert synergistic actions.     

鼠巨细胞病毒感染C57BL/6小鼠急性肝炎动物模型的建立与鉴定

目的：建立鼠巨细胞病毒（MCMV）感染C57BL/6小鼠急性肝炎模型并对其感染特点进行分析及鉴定。方法：将24 只C57BL/6小鼠随机分为阴性对照组（n =12）及病毒感染组（n =12），病毒感染组腹腔注射1.0×106 PFU（200 μL）MCMV悬液，阴性对照组注射等体积小鼠胚胎成纤维细胞（MEF）悬液。于感染后第3天和第7天取外周血分离血清检测谷丙转氨酶（ALT）及谷草转氨酶（AST）。同时进行肝组织病毒分离、组织病理学及MCMV IE和M55基因、细胞因子白细胞介素-6（IL-6）、白细胞介素-1β（IL-1β）、肿瘤坏死因子α（TNF-α）的检测。结果：病毒感染组肝组织匀浆病毒分离均为阳性，肝炎发生率为100%。在感染后第3天即发生肝炎病理改变，病毒感染组血清ALT及AST较阴性对照组明显升高（P ＜0.01）；病毒感染组肝脏HE染色第3天可见局灶性炎性细胞浸润及肝脏点灶状坏死，持续至第7天，Ishak评分较阴性对照组明显升高（P ＜0.01）；在感染后第3天病毒感染组肝组织内可检测到MCMV IE及M55基因，且在感染后第7天仍可测得IE基因；感染后第3天及第7天病毒感染组炎性细胞因子IL-6、TNF-α及IL-1β mRNA表达水平明显升高（P ＜0.05）。 结论：成功建立MCMV感染C57BL/6小鼠急性动物肝炎模型，其感染表现主要集中在急性感染前期。     

玻璃体内注射联合全身应用更昔洛韦治疗AIDS合并巨细胞病毒视网膜炎的临床疗效

目的　观察玻璃体内注射联合全身应用更昔洛韦治疗AIDS合并巨细胞病毒视网膜炎（cytomegalovirus retinitis,CMVR）的临床疗效。方法　收集2016年1月至2018年1月在广西医科大学第一附属医院收治的AIDS合并CMVR患者7例13眼。患者全身用药为更昔洛韦和膦甲酸钠注射液，更昔洛韦 5.0～7.5 mg·kg^-1，每天2次静脉滴注；成人每次给予膦甲酸钠3 g，每天3次静脉滴注；同时在局部玻璃体内注射更昔洛韦。治疗前后对比的指标有:最佳矫正视力、眼压、眼底照相情况、视觉诱发电位、眼电图和视网膜电图等检查；检测指标包括CD4+T细胞计数，血液、前房水、玻璃体CMV-DNA病毒载量等。通过检查结果辅助判断治疗效果。结果　6例患者治疗后最佳矫正视力为（0.72±0.83）logMAR，较治疗前（1.14±0.83）logMAR提高（P=0.001），其中1例患者经过全身和局部抗病毒治疗后视力不提高。患者治疗前后眼压均在正常范围内，治疗前为（13.62±3.04）mmHg（1 kPa=7.5 mmHg），治疗后为（12.77±2.89）mmHg，差异无统计学意义（P=0.119）。注药后患者眼底病变范围逐渐变小（P<0.05）。治疗1个月后，患者视觉诱发电位N2-P2振幅及眼电图光峰电位较治疗前升高（均为P<0.05），视觉诱发电位P2潜伏期和视网膜电图（明适应）a波及b波潜伏期和振幅治疗前后差异均无统计学意义（均为P>0.05）。3例患者治疗前血液中CMV-DNA病毒载量检测为阴性，1例患者2眼经过治疗后眼内液中CMV-DNA病毒载量检测为阴性。治疗前玻璃体CMV-DNA病毒载量均明显高于前房水，而前房水CMV-DNA病毒载量又明显高于血液（均为P<0.05），说明三个部位的CMV-DNA病毒载量从高到低的排列顺序为玻璃体>前房水>血液。治疗后玻璃体、前房水CMV-DNA病毒载量均低于治疗前，差异均有统计学意义（均为P<0.05）。治疗前后血液CMV-DNA病毒载量差异无统计学意义（P>0.05）。治疗前后CD4+T 细胞计数差异无统计学意义（P>0.05）。治疗前CD4+T细胞计数与眼内液、血液CMV-DNA病毒载量均呈负相关关系（均为P<0.05）；治疗过程中，CD4+T细胞计数与眼内液CMV-DNA病毒载量无相关关系（均为P>0.05），与血液CMV-DNA病毒载量呈负相关关系（P<0.05），但两者之间不存在直线回归关系（P>0.05）。治疗前及治疗过程中玻璃体CMV-DNA病毒载量与前房水CMV-DNA病毒载量均存在正相关关系（回归方程分别为:Y=20 178.973+0.165X，Y=171 849.77+0.168X，均为P<0.05）。所有患者术中和术后均未出现严重并发症。结论　静脉滴注抗病毒药物联合玻璃体内注射小剂量更昔洛韦是治疗AIDS合并CMVR安全有效的治疗方法。     

重症患者巨细胞病毒活动性感染与Th1/Th2型细胞因子的研究进展

重症患者巨细胞病毒(CMV)感染率极高,并且CMV感染后多呈潜伏性感染,同时感染CMV的重症患者往往缺乏典型的临床表现。但是当CMV再激活,即CMV活动性感染,此时可严重影响重症患者病情转归,导致多种不良预后的发生。究其机制为,CMV可通过影响辅助性T淋巴细胞1型和2型细胞(Th1/Th2)的功能,即通过调控Th1/Th2产生的细胞因子的数量及比例,来改变机体免疫状态,使CMV难以清除及易于再激活。因此Th1/Th2细胞因子的表达对CMV的再激活、复制和散播有着极其重要的作用及意义。本文就重症患者CMV活动性感染与Th1/Th2型细胞因子相互作用机制的研究进展作一综述。