Höfling K, Tracy S, Chapman N, Kim K S, Smith Leser J
Enterovirus Research Laboratory, Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68198-6495, USA.
J Virol. 2000 May;74(10):4570-8. doi: 10.1128/jvi.74.10.4570-4578.2000.
Group B coxsackieviruses (CVB) cause human myocarditis, while human adenovirus type 2 (Ad2) is implicated as an agent of this disease. The L1 loop of the Ad2 hexon protein has been demonstrated to be antigenic in rabbits. To evaluate the feasibility of a multivalent vaccine strain against the CVB and Ad2, we cloned the sequence encoding the Ad2 hexon L1 loop, flanked by dissimilar sequences encoding the protease 2A (2Apro) recognition sites, into the genome of an attenuated strain of CVB type 3 (CVB3/0) at the junction of 2Apro and the capsid protein 1D. Progeny virus (CVB3-PL2-Ad2L1) was obtained following transfection of the construct into HeLa cells. Replication of CVB3-PL2-Ad2L1 in diverse cell cultures demonstrated that the yield of the chimeric virus was between 0.5 to 2 log units less than the parental strain. Western blot analyses of the CVB3 capsid protein 1D in CVB3-PL2-Ad2L1-infected HeLa cells demonstrated production of the expected capsid protein. Viral proteins were detected earlier and in approximately fourfold greater amounts in CVB3-PL2-Ad2L1-infected HeLa cells than in CVB3/0-infected cells. Cleavage of the CVB3-PL2-Ad2L1 polyprotein by 2Apro was slowed, accompanied by an accumulation of the fusion 1D-L1 loop protein. Reverse transcription-PCR sequence analysis of CVB3-PL2-Ad2L1 RNA demonstrated that the Ad2 hexon polypeptide coding sequence was maintained in the chimeric viral genome through at least 10 passages in HeLa cells. Mice inoculated with CVB3-PL2-Ad2L1 demonstrated a brief viremia with no replication detectable in the heart but prolonged replication of virus in the pancreas in the absence of pathologic changes in either organ. CVB3-PL2-Ad2L1 induced binding and neutralizing anti-Ad2 antibodies, in addition to antibodies against CVB3 in mice. CVB3-PL2-Ad2L1 was used to challenge mice previously inoculated with CVB3/0 and with preexisting anti-CVB3 neutralizing-antibody titers; anti-Ad2 neutralizing and binding antibodies were induced in these mice at higher levels than in mice without anti-CVB3 immunity. The data demonstrate that a CVB vector can stably express an antigenic polypeptide of Ad2 from within the CVB open reading frame that results in the induction of protective immune responses against both viruses.
B组柯萨奇病毒（CVB）可引起人类心肌炎，而2型人腺病毒（Ad2）被认为是该疾病的病原体。已证明Ad2六邻体蛋白的L1环在兔子中具有抗原性。为了评估一种针对CVB和Ad2的多价疫苗株的可行性，我们将编码Ad2六邻体L1环的序列（两侧为编码蛋白酶2A（2Apro）识别位点的不同序列）克隆到3型柯萨奇病毒减毒株（CVB3/0）的基因组中，位于2Apro和衣壳蛋白1D的连接处。将构建体转染到HeLa细胞后获得了子代病毒（CVB3-PL2-Ad2L1）。CVB3-PL2-Ad2L1在多种细胞培养物中的复制表明，嵌合病毒的产量比亲代毒株低0.5至2个对数单位。对CVB3-PL2-Ad2L1感染的HeLa细胞中CVB3衣壳蛋白1D的蛋白质免疫印迹分析表明产生了预期的衣壳蛋白。在CVB3-PL2-Ad2L1感染的HeLa细胞中比在CVB3/0感染的细胞中更早且检测到的病毒蛋白量大约多四倍。2Apro对CVB3-PL2-Ad2L1多聚蛋白的切割减慢，伴随着融合的1D-L1环蛋白的积累。对CVB3-PL2-Ad2L1 RNA的逆转录-PCR序列分析表明，Ad2六邻体多肽编码序列在HeLa细胞中至少传代10次后仍保留在嵌合病毒基因组中。接种CVB3-PL2-Ad2L1的小鼠出现短暂病毒血症，心脏中未检测到复制，但病毒在胰腺中持续复制，而两个器官均无病理变化。CVB3-PL2-Ad2L1除了诱导小鼠产生针对CVB3的抗体外，还诱导产生结合和中和抗Ad2抗体。用CVB3-PL2-Ad2L1对先前接种过CVB3/0且已有抗CVB3中和抗体滴度的小鼠进行攻击；在这些小鼠中诱导产生的抗Ad2中和和结合抗体水平高于没有抗CVB3免疫力的小鼠。数据表明，CVB载体可以在CVB开放阅读框内稳定表达Ad2的抗原多肽，从而诱导针对两种病毒的保护性免疫反应。
