The third variable (V3) loop of HIV-1 gp120 is an immunodominant region targeted by neutralizing antibodies (nAbs). Despite limited breadth, better characterization of the structural details of the interactions between these nAbs and their target epitopes would enhance our understanding of the mechanism of neutralization and facilitate designing better immunogens to induce nAbs with greater breadth. Recently, we isolated two anti-V3 neutralizing monoclonal antibodies (MAbs), 10A3 and 10A37, from a rabbit immunized with gp120 of the M group consensus sequence. In this study, crystal structures of these MAbs bound to target epitopes were determined. 10A3 binds to the V3 crown (³⁰³TRKSIHIGPGRAF³¹⁷) using the cradle binding mode, similar to human V3 MAbs encoded by IGHV5-51 germ line genes, and its epitope structure resembles that bound to the human antibodies. In contrast, 10A37, which exhibits greater breadth and potency than 10A3, binds the V3 crown and the succeeding stem region (³⁰⁸HIGPGRAFYTTGEI³²³). Unexpectedly, the ³¹⁵RAFYTT³²⁰ portion of the epitope existed as helical turns, a V3 structure that has not been observed previously. Its main chain-dominated antigen-antibody interactions not only explain the broad neutralization of 10A37 but also show that its epitope is a potential vaccine target to be further evaluated. In conclusion, our study provides novel insights about neutralization-susceptible epitope structures of the V3 loop of HIV-1 gp120 and demonstrates that, despite low amino acid sequence similarity to human antibody germ line genes, rabbits can serve as a useful animal model to evaluate human vaccine candidates.

IMPORTANCE The apex crown of V3 of HIV-1 gp120 is the most immunogenic region of the surface glycoprotein, and many MAbs targeting this region have been developed. Structural understanding of V3 crown MAbs not only can help understand how antibody responses target this unique region but also contribute to immunogen design for vaccine development. We present here crystal structures of two neutralizing V3 MAbs, 10A3 and 10A37, developed from a rabbit immunized with gp120. Our analysis of 10A3 in complex with V3 provided a detailed example of how epitope complexity can evolve with affinity maturation, while that of 10A37 revealed a novel V3 binding mode targeting the C-terminal side of the V3 crown and showed that this region can form a helical structure. Our study provides novel insights about neutralization-susceptible V3 epitope structures and demonstrates that rabbits can serve as a useful animal model to evaluate human vaccine candidates.

HIV-1 gp120的第三个变量（V3）环是中和抗体（nAbs）靶向的免疫优势区域。尽管广度有限，但是更好地表征这些nAb与目标表位之间相互作用的结构细节将增强我们对中和机理的理解，并有助于设计更好的免疫原来诱导更大广度的nAb。最近，我们从M组共有序列的gp120免疫的兔子中分离了两种抗V3中和性单克隆抗体（MAb）10A3和10A37。在这项研究中，确定了与目标表位结合的这些单克隆抗体的晶体结构。10A3绑定到V3冠（³⁰³ TRKSIHIGPGRAF ³¹⁷）使用摇篮结合模式，类似于IGHV5-51种系基因编码的人V3 MAb，其表位结构类似于与人抗体结合的表位结构。相反，表现出比10A3更大的广度和效力的10A37将V3冠和随后的茎区域（³⁰⁸ HIGPGRAFYTTGEI ³²³）结合在一起。出乎意料的是³¹⁵ RAFYTT ³²⁰表位的一部分呈螺旋状转弯，这是以前未曾观察到的V3结构。它的主链占主导地位的抗原-抗体相互作用不仅解释了10A37的广泛中和作用，还表明其表位是潜在的疫苗靶标，需要进一步评估。总之，我们的研究提供了有关HIV-1 gp120 V3环中和敏感性表位结构的新颖见解，并表明，尽管与人抗体种系基因的氨基酸序列相似性低，兔子仍可作为评估动物的有用动物模型人类疫苗候选者。

重要性HIV-1 gp120的V3的最高冠是表面糖蛋白的最具免疫原性的区域，并且已开发出许多靶向该区域的单抗。对V3冠状单抗的结构理解不仅可以帮助理解抗体应答如何靶向该独特区域，而且还有助于疫苗开发的免疫原设计。我们在这里介绍了两种中和V3单抗10A3和10A37的晶体结构，这些晶体是从用gp120免疫的兔体内发育而来的。我们对与V3结合的10A3的分析提供了表位复杂性如何随着亲和力成熟而演化的详细示例，而对10A37的分析则揭示了针对V3冠的C端侧的新型V3结合模式，并表明该区域可以形成一个螺旋结构。