Hamad Abdelrahman S, Edward Eva A, Sheta Eman, Aboushleib Hamida M, Bahey-El-Din Mohammed
Department of Microbiology and Immunology, Faculty of Pharmacy, Alexandria University, Alexandria P.O. Box 25435, Egypt.
Pathology Department, Faculty of Medicine, Alexandria University, Alexandria P.O. Box 21131, Egypt.
Vaccines (Basel). 2022 Dec 23;11(1):28. doi: 10.3390/vaccines11010028.
(PA) is a Gram-negative pathogen responsible for fatal nosocomial infections worldwide. Iron is essential for Gram-negative bacteria to establish an infection. Therefore, iron acquisition proteins (IAPs) of bacteria are attractive vaccine targets.
A "Reverse Vaccinology" approach was employed in the current study. Expression levels of 37 IAPs in various types of PA infections were analyzed in seven previously published studies. The IAP vaccine candidate was selected based on multiple criteria, including a high level of expression, high antigenicity, solubility, and conservation among PA strains, utilizing suitable bioinformatics analysis tools. The selected IAP candidate was recombinantly expressed in and purified using metal affinity chromatography. It was further evaluated in vivo for protection efficacy. The novel immune adjuvant, naloxone (NAL), was used.
HasAp antigen met all the in silico selection criteria, being highly antigenic, soluble, and conserved. In addition, it was the most highly expressed IAP in terms of average fold change compared to control. Although HasAp did excel in the in silico evaluation, subcutaneous immunization with recombinant HasAp alone or recombinant HasAp plus NAL (HasAP-NAL) did not provide the expected protection compared to controls. Immunized mice showed a low IgG2a/IgG1 ratio, indicating a T-helper type 2 (Th2)-oriented immune response that is suboptimal for protection against PA infections. Surprisingly, the bacterial count in livers of both NAL- and HasAp-NAL-immunized mice was significantly lower than the count in the HasAp and saline groups. The same trend was observed in kidneys and lungs obtained from these groups, although the difference was not significant. Such protection could be attributed to the enhancement of innate immunity by NAL.
We provided a detailed in silico analysis of IAPs of PA followed by in vivo evaluation of the best IAP, HasAp. Despite the promising in silico results, HasAp did not provide the anticipated vaccine efficacy. HasAp should be further evaluated as a vaccine candidate through varying the immunization regimens, models of infection, and immunoadjuvants. Combination with other IAPs might also improve vaccination efficacy. We also shed light on several highly expressed promising IAPs whose efficacy as vaccine candidates is worthy of further investigation.
铜绿假单胞菌（PA）是一种革兰氏阴性病原体，在全球范围内可导致致命的医院感染。铁对于革兰氏阴性菌建立感染至关重要。因此，细菌的铁获取蛋白（IAPs）是有吸引力的疫苗靶点。
本研究采用了“反向疫苗学”方法。在七项先前发表的研究中分析了37种IAPs在各种类型PA感染中的表达水平。利用合适的生物信息学分析工具，基于多种标准选择IAP疫苗候选物，包括高表达水平、高抗原性、溶解性以及在PA菌株中的保守性。所选的IAP候选物在大肠杆菌中重组表达并使用金属亲和层析进行纯化。在体内进一步评估其保护效果。使用了新型免疫佐剂纳洛酮（NAL）。
HasAp抗原符合所有计算机模拟选择标准，具有高抗原性、溶解性且保守。此外，就与对照相比的平均变化倍数而言，它是表达最高的IAP。尽管HasAp在计算机模拟评估中表现出色，但与对照组相比，单独皮下注射重组HasAp或重组HasAp加NAL（HasAP-NAL）并未提供预期的保护。免疫小鼠显示出低IgG2a/IgG1比率，表明以2型辅助性T细胞（Th2）为主导的免疫反应，这种反应对于抵抗PA感染并非最佳。令人惊讶的是，NAL免疫组和HasAp-NAL免疫组小鼠肝脏中的细菌计数均显著低于HasAp组和生理盐水组。在这些组的肾脏和肺中也观察到相同趋势，尽管差异不显著。这种保护可能归因于NAL对固有免疫的增强作用。
我们对PA的IAPs进行了详细的计算机模拟分析，随后对最佳IAP HasAp进行了体内评估。尽管计算机模拟结果很有前景，但HasAp并未提供预期的疫苗效力。应通过改变免疫方案、感染模型和免疫佐剂，将HasAp作为疫苗候选物进一步评估。与其他IAPs联合使用也可能提高疫苗效力。我们还揭示了几种高表达的有前景的IAPs，其作为疫苗候选物的效力值得进一步研究。
