Recent studies have revealed that the interface of gp120 and gp41 and some parts of gp41 are also critical epitopes for elicitation of broadly neutralizing antibodies. Therefore, potential trimeric gp41 or gp140 immunogen candidates are needed. Previously, we developed a trimer motif MTQ and demonstrated that it could help formation of trimeric gp120 and gp140 proteins. In the present study, we immunized Balb/c mice using trimeric gp41-expressing plasmid for prime and monomeric gp41 or trimeric gp140 protein as well as a mutant (Q577A) for boost. The antibody responses in the context of regimens with various immunization intervals and DNA adjuvants including praziquantel (PZQ), cimetidine (CIM), and amiloride (AML) were evaluated. We found that these three adjuvants were not enough to elicit remarkable specific Abs after gp41 DNA immunization, while AML could significantly promote humoral immune responses after protein boosts. Long immunization interval could induce the specific binding Abs earlier and higher and maintain a high level of Abs in the following 27 weeks after final protein boost. Moreover, two times of protein boosts with DNA adjuvant and a longer time interval achieved a higher titer of specific Abs than three times of protein boosts with a shorter time interval. Q577A mutant was benefit for trimeric gp140 boost in the production of binding Abs but harmful to inducing neutralizing Abs, while this mutant in monomeric gp41 presented the opposite trend which may be associated with the immunogen structures. This study highlights the significance of DNA adjuvant Amiloride and long immunization interval in promoting antibody responses and provides new insights into effective HIV immunization regimen design in the future.

最近的研究表明，gp120和gp41以及gp41的某些部分的界面也是引发广泛中和抗体的关键表位。因此，需要潜在的三聚体gp41或gp140免疫原候选物。以前，我们开发了三聚体基序MTQ，并证明它可以帮助形成三聚体gp120和gp140蛋白。在本研究中，我们使用表达三聚体gp41的质粒对初生和单体gp41或三聚体gp140蛋白以及突变体（Q577A）进行免疫，以免疫Balb / c小鼠。在具有各种免疫间隔的方案和DNA佐剂（包括吡喹酮（PZQ），西咪替丁（CIM）和阿米洛利（AML））中评估了抗体应答。我们发现这三种佐剂不足以在gp41 DNA免疫后引发明显的特异性Abs，而AML可以在蛋白质增强后显着促进体液免疫反应。较长的免疫间隔可在最终蛋白质加强后的27周内更早和更高地诱导特异性结合Abs，并维持高水平的Abs。而且，与使用较短时间间隔的三倍蛋白质增强相比，使用DNA佐剂增强蛋白质的两倍和更长的时间间隔可实现更高的特异性Abs效价。Q577A突变体有利于三聚体gp140增强结合Abs的产生，但对诱导中和Abs有害，而单体gp41中的此突变体则呈现相反的趋势，可能与免疫原结构有关。