Differential gene expression in W614A-3S peptide-specific B cell populations following W614A-3S peptide-KLH vaccination using Squalene emulsion and Alum formulation. Differential gene expression in W614A-3S peptide-specific B cell populations following W614A-3S peptide-KLH vaccination using Squalene emulsion and Alum formulation

W614A-3S peptide is a modified 3S motif of the HIV-gp41 (mutation W614A). We previously detected the presence of natural neutralizing antibodies directed against W614A-3S peptide (NAbs) in long-term non-progressor HIV+ patients. Here, we compared the efficacy of W614A-3S peptide formulated in either squalene emulsion (SQE) or in aluminum hydroxide (Alum) in inducing broadly-NAbs (bNAbs). Rabbit and mouse models were used to screen the induction of bNAbs following 4 immunizations. SQE was more efficient than Alum formulation in inducing W614A-3S-specific bNAbs with up to 67-93% of HIV strains neutralized. We then analyzed the quality of peptide-specific murine B cells by single-cell gene expression by quantitative Reverse Transcription-PCR and single-cell V(D)J sequencing. We found more frequent germinal center B cells in SQE than in Alum, albeit with a different gene expression profile. The V(D)J sequencing of W614A-3S-specific BCR showed significant differences in BCR sequences and validates the dichotomy between adjuvant formulations. All sixteen BCR sequences which were cloned were specific of peptide. Adjuvant formulations of W614A-3S-peptide-conjugated immunogen impact the quantity and quality of B cell immune responses at both the gene expression level and BCR sequence. We used a single-cell quantitative Reverse Transcription-PCR (qRT-PCR) approach to compare between the two formulations the quality of W614A-3S-specific B cell populations isolated from draining lymph nodes, one week after 2nd and 3rd immunizations (W3 and W5, respectively). Overall design: Draining lymph nodes (dLNs; n=pool of 5 mice/condition - two independent experiments) were harvested at W3 and W5 after immunization, and used to isolate peptide-specific B cells and to perform single-cell gene expression analysis.

W614A-3S肽（W614A-3S peptide）是携带W614A突变的人类免疫缺陷病毒包膜蛋白41（HIV-gp41）3S基序的修饰型肽段。本研究团队此前已在长期非进展性HIV阳性患者体内，检测到靶向W614A-3S肽的天然中和抗体（neutralizing antibodies, NAbs）。本研究对比了以角鲨烯乳剂（squalene emulsion, SQE）或氢氧化铝（aluminum hydroxide, Alum）为佐剂的W614A-3S肽制剂诱导广谱中和抗体（broadly neutralizing antibodies, bNAbs）的效果。研究采用家兔与小鼠模型，通过4次免疫程序筛选广谱中和抗体的诱导情况，结果显示，相较于氢氧化铝佐剂制剂，角鲨烯乳剂佐剂制剂诱导W614A-3S肽特异性广谱中和抗体的效率更高，可中和67%~93%的HIV毒株。随后，本研究通过定量反转录聚合酶链反应（quantitative Reverse Transcription-PCR, qRT-PCR）单细胞基因表达分析与单细胞V(D)J测序技术，对肽特异性小鼠B细胞的质量特征进行分析：相较于氢氧化铝佐剂组，角鲨烯乳剂组的生发中心B细胞比例更高，尽管二者的基因表达谱存在差异。对W614A-3S肽特异性B细胞受体（B cell receptor, BCR）的V(D)J测序结果显示，BCR序列存在显著差异，验证了两种佐剂制剂之间的免疫应答分化特征。所有被克隆的16条BCR序列均对该肽段具有特异性。W614A-3S肽偶联免疫原的佐剂制剂，可从基因表达水平与BCR序列层面同时影响B细胞免疫应答的数量与质量。本研究采用单细胞定量反转录聚合酶链反应方法，对比了两种佐剂制剂在第二次与第三次免疫后1周（分别记为免疫后第3周W3和第5周W5），从引流淋巴结中分离得到的W614A-3S肽特异性B细胞群体的质量差异。总体实验设计：分别于免疫后第3周和第5周采集引流淋巴结（draining lymph nodes, dLNs；每组为5只小鼠的淋巴结混合样本，共开展2次独立重复实验），用于分离肽特异性B细胞并进行单细胞基因表达分析。