扇贝、对虾基因组BAC文库构建及其重要功能基因的初步筛选和分析

基因组大片段BAC文库是进行生物遗传学和基因组学研究必不可少的基础工具。为了深入开展栉孔扇贝（Chlamys farreri）和凡纳滨对虾（Litopenaeus vannamei）基因组学研究、阐明其基因组的结构与功能、图位克隆重要功能基因、构建高密度物理图谱并最终实现与已有遗传连锁图的整合，本研究在植物基因组BAC文库构建技术的基础上，针对海洋生物的特点进行了大胆的改革与尝试，最终成功构建了C. ferrari和L. vannamei两种重要海水养殖动物的基因组BAC文库。 本文构建的栉孔扇贝基因组BAC文库，由BamHI文库和MboI文库构成。其中BamHI文库含有73,728个BAC克隆，空载率约为1%，平均插入片段约为110 kb，覆盖栉孔扇贝单倍体基因组约8倍；MboI文库共有7,680个克隆组成，平均插入片段大小约为145 kb，插入率为100%，覆盖栉孔扇贝单倍体基因组约1.1倍。两个栉孔扇贝基因组BAC文库共由81,408个克隆组成，平均插入片段约为113 kb，覆盖率约为栉孔扇贝单倍体基因组大小的9.1倍。 将栉孔扇贝基因组BAC文库的192个384微孔培养板中的73,728个BAC克隆以4 x 4点阵形式制备了高密度DNA薄膜，用于对感兴趣的基因及DNA序列的筛选。高密度DNA薄膜的覆盖率约为栉孔扇贝单倍体基因组的8.3倍。针对栉孔扇贝先天免疫系统通路的6个重要功能基因，根据栉孔扇贝cDNA序列以及异缘物种DNA序列设计了Overgo探针。利用Overgo探针对高密度DNA薄膜杂交筛选的结果显示，平均每个基因检测到7.3个潜在阳性克隆。 本研究所构建的凡纳滨对虾基因组HindIII酶切BAC文库共有102,528个BAC克隆，存放于267个384微孔培养板中，平均插入片段大小约为101 kb，空载率约为5%，覆盖L. vannamei单倍体基因组约5倍。将其中240个384微孔培养板中的92,160个BAC克隆以4 x 4的矩阵排列形式制作了5张高密度凡纳滨对虾DNA薄膜，约覆盖整个对虾单倍体基因组的4.5倍。针对6个与对虾免疫、生殖生理有关的重要功能基因设计了Overgo探针，杂交筛选出20个阳性克隆，平均每个基因有3.3个潜在阳性克隆。 以上筛选结果不仅为进一步研究这些功能基因的结构与功能、表达与调控，揭示它们在扇贝和对虾以及其他近缘种的免疫系统、抗逆和生殖生理过程中的作用机理打下了基础，同时也间接验证了栉孔扇贝和凡纳滨对虾基因组BAC文库将成为基因筛选、基因的结构与功能分析、基因图位克隆、物理图谱构建以及大规模全基因组测序等方面的有力工具。

Preliminary study of the dynamic origin of the distribution pattern of bottom sediments on the continental shelves of the Bohai Sea, Yellow Sea and East China Sea

The bottom sediment types in the Bohai Sea, Yellow Sea and East China Sea (BYECS) are diversified, and their distribution pattern is very complicated. However, the bottom sediment types can be simplified to be sandy sediment, clayey sediment and mixed sediment, which comprise the complicated distribution pattern of bottom sediment in the BYECS. The continental shelves of the BYECS are broad, with shallow water depths and tidal currents which are permanent and dominate the marine dynamics in the BYECS. Based on numerical simulation of tidal elevations and currents in the BYECS, the rates of suspended load transport and bed load transport during a single tidal cycle for sediments of eight different grain size ranges are calculated. The results show that any sediment, whose threshold velocity is less than that of tidal current, has the same transport trend. Suspended load transport rare, bed load transport rate, and the ratio of the former to the latter decrease with grain size becoming coarser and coarser. The erosion/accretion patterns of sediments with different grain sizes are determined by the sediment transport rate divergences, and the results show that the patterns are the same for sediments with different grain sizes. Three main bottom sediment types, i.e. sandy sediment mainly composed of fine sand, clayey sediment mainly composed of silty clay, and mixed sediment mainly composed of fine sand, silt, and clay, are obtained by computation. The three bottom sediment types and their distribution pattern are consistent not only with sediment transport field and the sea bed erosion/accretion pattern obtained by simulation, but also with field data of bottom sediment types and divisions. In the BYECS, sand ridges form mainly in the areas with strong rectilinear tidal currents, sand sheets form mainly in the areas dominated by strong rotatory tidal currents, and clayey sediments, i.e. mud patches, form mainly in the areas with weak tidal currents. Hence, not only the sandy sediments but also the clayey sediments in the BYECS are formed under the control of the whole tidal current field of the BYECS. The three main bottom sediment types are not isolated respectively-in fact, they constitute a whole tidal depositional system. Under the condition with no cyclonic cold eddy, the clayey sediments in the BYECS can form in weak tidal current environments. Therefore, a cold eddy is not necessary for the deposition of clayey sediments in the BYECS. (C) 2000 Academic Press.

The unique skeleton of siliceous sponges (Porifera; Hexactinellida and Demospongiae) that evolved first from the Urmetazoa during the Proterozoic: a review

Sponges (phylum Porifera) had been considered as an enigmatic phylum, prior to the analysis of their genetic repertoire/tool kit. Already with the isolation of the first adhesion molecule, galectin, it became clear that the sequences of sponge cell surface receptors and of molecules forming the intracellular signal transduction pathways triggered by them, share high similarity with those identified in other metazoan phyla. These studies demonstrated that all metazoan phyla, including Porifera, originate from one common ancestor, the Urmetazoa. The sponges evolved prior to the Ediacaran-Cambrian boundary (542 million years ago [myr]) during two major "snowball earth events", the Sturtian glaciation (710 to 680 myr) and the Varanger-Marinoan ice ages (605 to 585 myr). During this period the ocean was richer in silica due to the silicate weathering. The oldest sponge fossils (Hexactinellida) have been described from Australia, China and Mongolia and are thought to have existed coeval with the diverse Ediacara fauna. Only little younger are the fossils discovered in the Sansha section in Hunan (Early Cambrian; China). It has been proposed that only the sponges possessed the genetic repertoire to cope with the adverse conditions, e.g. temperature-protection molecules or proteins protecting them against ultraviolet radiation. The skeletal elements of the Hexactinellida (model organisms Monorhaphis chuni and Monorhaphis intermedia or Hyalonema sieboldi) and Demospongiae (models Suberites domuncula and Geodia cydonium), the spicules, are formed enzymatically by the anabolic enzyme silicatein and the catabolic enzyme silicase. Both, the spicules of Hexactinellida and of Demospongiae, comprise a central axial canal and an axial filament which harbors the silicatein. After intracellular formation of the first lamella around the channel and the subsequent extracellular apposition of further lamellae the spicules are completed in a net formed of collagen fibers. The data summarized here substantiate that with the finding of silicatein a new aera in the field of bio/inorganic chemistry started. For the first time strategies could be formulated and experimentally proven that allow the formation/synthesis of inorganic structures by organic molecules. These findings are not only of importance for the further understanding of basic pathways in the body plan formation of sponges but also of eminent importance for applied/commercial processes in a sustainable use of biomolecules for novel bio/inorganic materials.

大黄属三种植物不同部分提取物清除羟基自由基的体外实验研究

利用Fenton反应产生的羟基自由基,采用比色法对大黄属药用植物:唐古特大黄、波叶大黄、穗序大黄不同部分提取液清除羟基自由基的活性进行了研究,结果表明:三种植物不同部分的提取液均有一定的清除羟基自由基的能力,清除能力因种、植株部分和提取方法的不同而异.三种植物中清除率最高的部分分别是:唐古特大黄根及根茎的水提液,为79.0%;波叶大黄叶片的乙醇提取液,为84.5%;穗序大黄叶片水提液和叶柄的乙醇提取液,分别为70.1%和70.7%.正品大黄植株地下部分清除率较非正品高,但地上部分清除率却低于非正品.

基于改进角度直方图和最小二乘法的移动机器人地图构建

本文主要研究完全未知结构化环境下的移动机器人二维几何地图构建及其不确定性描述问题。在考虑测量噪声干扰的基础上，基于改进的角度直方图算法进行环境线段特征提取和参数初始估计，然后利用加权最小二乘法对线段特征参数及其方差进行精确估计，并同时给出了多位姿地图合并的处理方法.文章最后给出了在SmartROB2机器人平台上进行的实验结果，证明了算法的有效性和实用性。

北天山新生代构造演化与古环境演变

The Tianshan Mountains is located about 1000-2000 km north of the India-Asia suture and is the most outstanding topography in central Asia, with transmeridional length of nearly 2500 km, north-southern wideness of ~ 300-500 km, peaks exceeding 7000 m above sea level (asl.), and average altitude of over 4000 m asl. Much of the modern relief of the Tianshan Range is a result of contraction driven by the collision of the India subcontinent with the southern margin of Asia, which began in early Tertiary and continues today. Understanding where, when and how the deformation of the Tianshan Mountains occurred is essential to decipher the mechanism of intracontinental tectonics, the process of foreland basin evolution and mountain building, and the history of climate change in central Asia. In order to better constrain the Cenozoic building history of the Tianshan Mountains and the climate change in the southern margin of the Junggar Basin, we carried out multiple studies of magnetostratigraphy, sedimentology, and stable isotopes of paleosol carbonate at the Jingou River section, which is located at the Huoerguosi anticline, the westernest one of the second folds and thrust faults zone in the northern piedmont of the Tianshan Mountains. The Jingou River section with a thickness of about 4160 m is continuous in deposits according to the observed gradual change in sedimentary environments and can be divided into five formations: Anjihaihe, Shawan, Taxihe, Dushanzi and Xiyu in upward sequence. Characteristic remamences were isolated by progressive thermal demagnetization, generally between 300 and 680℃. A total of 1133 out of 1607 samples yielded well-defined ChRMs and were used to establish the magnetostratigraphic column of a 3270-m-thick section from the exposed base of the Anjihaihe Formation to the middle of the Xiyu Formation. Two vertebrate fossil sites and a good correlation with the CK95 geomagnetic polarity time scale suggest that the section was deposited from ~30.5 to ~4.6 Ma and the age of the top of the Xiyu formation is ~2.6 Ma based on an extrapolation of the sedimentation rates. A plot of magnetostratigraphic age vs. height at the Jingou River section shows that significant increases in sedimentation rates as well as notable changes in depositional environments occurred at ~26-22.5 Ma, ~13-11 Ma and ~7 Ma, which represent the initial uplift of the Tianshan Mountains and two subsequent rapid uplift events. In addition, changes in sedimentation rates display characteristic alternations between increases and decreases, which probably indicate that the uplift of the Tianshan Mountains was episodic. We discussed the history of C4 biomass and climatic conditions in the southern margin of the Junggur Basin using the stable carbon and oxygen isotope composition of paleosol carbonates from the Jingou River section during ~17.5-6.5 Ma. The δ13C values indicate that the proportion of C4 biomass was uniform and moderate (15-20 %) during the interval of ~17.5-6.5 Ma. We proposed three hypotheses for this pattern of C4 biomass: (1) counteraction of two opposed factors (global cooling since ~15 Ma and thereafter increased dry and seasonality in central Asia) controlling the growth of C4 grasses, (2) variability in abundance of C3 grasses relative to C3 trees and shrubs if vegetation had ever changed in ecosystems, and (3) the higher latitude of the studied region. The δ18O values show a stepwise negative trend since ~13 Ma which may be attributed to three factors: (1) the temperature decreasing gradually after the middle Miocene (~15 Ma), (2) the increasing contribution of the moistures carried by the polar air masses from the Arctic Ocean to precipitation, and (3) the gradual retreat westward and disappearance of the Paratethys Ocean. Among them, which one played a more important role will need further study of the paleoclimate in central Asia.

五台山区义兴寨岩体、车厂－北台岩体地球化学和变质作用及其大地构造意义

There are many Archean TTG grey suites in the Wutaishan area, northern Shanxi Province, China. In the past one hundred years, many geologists have done excellent research work in the Wutaishan and its adjacent regions. However, the TTG suites were almost neglected. Located in the northern slope of Mt. Hengshan-namely the Archean Hengshan Island Arc, intruded the Zhujiafang supercrustal rocks at almost 2.5Ga, the Yixingzhai TTG Suite is originated from partial melting of the ancient lower crust upper mantle by REE and trace elements, and the emplacement occurred in an Archean island arc. The rocks are mainly of tonalitic, I type, and calc-alkaline trends are found in the magmatic evolution. At almost 1.8 Ga, the suite was transformed to be dome-like schists in an arc-arc collision event, and the rocks were metamorphosed to an extent of amphibolitic to granulitic facies. The peak metamorphic condition is of 710-760 ℃/0.68-0.72GPa, and the subsequent cooling history is recorded as 560-620 ℃/0.46-0.60GPa. In the center of the Mt. Wutaishan-known as the Archean Wutaishan Island Arc, intruded the Archean Chechang-Beitai TTG Suite, which is of 2.5Ga old and of trondhjemitic and tonalitic, with coexisting I- and S-types and a trondhjemitic magmatic evolution trend. Through REE and trace elements, the suite is believed to be from the partial melting of the ancient lower crust or upper mantle. The 1.8 Ga collision event also made the suite gneissic and the it was metamorphosed to be amphibolitic facies, whose peak condition is approximately of 680 (±50) ℃/0.7Gpa, and the subsequent cooling process is recorded as 680 (±50) ℃、550(±50) ℃、420(±10) ℃. Crustal growth is fulfilled through magmatic intrusion as well as eruption at about 2.5Ga, arc-arc collision at about 1.8 Ga in the Wutaishan area and its environs. Additionally, the biotite-muscovite and muscovite-plagioclase geothermometers are refined, and the biotite-hornblende geothermometer is developed in this dissertation.

论云南会泽富铅锌矿床成矿模式

通过构造地质学、矿床学、地球化学研究，认为燕山早期环太平洋板块的俯冲碰撞与小江深断裂带、曲靖－昭通隐伏断裂带的左行走滑运动为深源流体的形成和贯入提供了有利的构造背景，成矿物质主要来源于深源流体和蒸发岩层，矿体本身是富含铅锌锗等的成矿流体沿构造贯入的产物，矿床定位直接受北东构造带控制。在此基础上，提出“深源流体贯入－蒸发岩层萃取－构造控制”的成矿模式。

可见光响应光解水制氢的半导体光催化剂

研究开发可见光响应的光催化剂一直是光解水制氢的首要目标。近年来通过能带调控等手段实现光催化剂的可见光化被广泛研究，并取得了令人注目的进展。本文综述了通过能带调变实现可见光化的各种手段，包括TiO2掺杂特别是阴离子掺杂、能响应可见光的新型固溶体和单相光催化材料的开发以及Z-型反应系统的构筑，以及通过电子结构的分析阐述其可见光化的机理.

芙蓉锡矿田骑田岭花岗岩黑云母矿物化学组成及其对锡成矿的指示意义

芙蓉锡矿田骑田岭复式岩体主要由早阶段角闪石黑云母花岗岩和晚阶段黑云母花岗岩组成.电子探针分析结果表明角闪石黑云母花岗岩中的黑云母属于铁黑云母,黑云母花岗岩中的黑云母属于铁叶云母.相对于黑云母花岗岩,角闪石黑云母花岗岩中黑云母的MgO、TiO2含量偏高,Al2O3含量偏低.矿物化学研究结果显示,角闪石黑云母花岗岩中黑云母的结晶温度、氧逸度(logfO2)分别为680℃～740℃、-16.00～-15.31,黑云母花岗岩中黑云母的结晶温度、氧逸度分别为530℃～650℃、-19.20～-17.50.从角闪石黑云母花岗岩到黑云母花岗岩,岩浆结晶温度和氧逸度逐渐降低.与花岗岩有关的共存流体性质的研究发现,与角闪石黑云母花岗岩共存的热液流体log(fH2O/fHF)fluid,log(fH2O/fHCl)fluid,log(fHF/fHCl)fiuid值分别为4.22～4.39,2.78～3.24,-1.82～-1.73,而与黑云母花岗岩共存的热液流体log(fH2O/fHF)fluid,log(fH2OfHCl)fluid,log(fHF/fHCl)fluid值分别为3.27～3.53,2.85～3.22,-0.75～-0.22,可见与两种岩石类型共存热液流体的性质存在明显差异,且热液中Cl、Sn含量变化与岩浆结晶分异指数呈正相关关系.骑田岭岩体从角闪石黑云母花岗岩到黑云母花岗岩,随着岩浆的演化.岩浆结晶期后分异出的热液流体向富Cl和Sn方向演化.芙蓉锡矿田的成矿流体应主要来源于黑云母花岗岩岩浆结晶期后分异出的岩浆热液.

甘肃阳山金矿同位素地球化学特征及矿床成因

甘肃文县阳山金矿的探明黄金储量已达308t，平均品位4.74g/t，是我国地质勘查储量最大的金矿床。该矿床产于西秦岭造山带，是一个同碰撞形成的类卡林型金矿床，矿体受EW向韧脆性剪切带控制，赋矿围岩为泥盆系碳质千枚岩-板岩-碳酸盐-硅质岩和侵入其中的花岗斑岩脉。流体成矿过程包括：形成石英-绢云母-黄铁矿组合的早阶段，形成石英-黄铁矿-毒砂-方铅矿等多金属组合的主成矿阶段，形成碳酸盐-辉锑矿-石英网脉的晚阶段。 与矿体关系较为密切的花岗斑岩富集LILE 和 LREE, 亏损 Ba, Sr, Nb, Ta, P 和Ti，ΣREE＝54.35～124.01 μg/g ，(La/Yb)N=9.72～27.80，δEu=0.70～0.89， ISr值为0.70806～0.71756，平均0.71107；εNd(t)平均-3.4；Nd模式年龄（T2DM）平均1.34（Ga）。表明花岗斑岩岩浆应源自成熟度较低的中元古代基底地壳物质。花岗斑岩的(206Pb/204Pb)220Ma、(207Pb/204Pb)220Ma和(208Pb/204Pb)220Ma的平均值分别为17.875、15.604和38.296，与秦岭微陆块的中元古代基底和碧口地体碧口群的Pb同位素组成一致。考虑到前人获得碧口群的年龄为1.235～1.367Ga，而秦岭微陆块沿勉略缝合带向南仰冲到碧口地体之上，我们认为由碧口群等组成的俯冲板片的变质脱水熔融作用导致了阳山金矿带花岗斑岩的形成。因此，阳山金矿带的花岗斑岩是扬子与华北大陆中生代碰撞造山过程中形成的同碰撞花岗岩类。 最新的S，Sr和Pb同位素研究表明：热液成矿早阶段的黄铁矿的34S值范围介于-15.5‰~6.59‰之间，总体离散性比较大，显示沉积地层来源的特征，硫同位素组成属离散型，不具有岩浆主导的成矿的塔式效应。花岗岩中黄铁矿硫同位素范围很集中，34S值处于-1.47‰～2.12‰之间，本区花岗斑岩不可能为成矿物质的主要来源。矿石硫化物的初始锶同位素比值范围较大（0.70877～0.71697，平均为0.71258），显示成矿物质并非单一来源，考虑到花岗斑岩先于矿床形成，只在后期构造作用的岩体部分成矿的地质事实，少量矿石中的低锶同位素比值黄铁矿有可能来自作为围岩的花岗斑岩，也可能来自基底物质。矿石硫化物Pb同位素206Pb/204Pb=17.552～18.853，平均18.260；207Pb/204Pb=15.574～15.928，平均15.685；208Pb/204Pb=37.894～39.293，平均38.680，变化范围比较大。μ=9.46~10.06，平均为9.65，ω值介于36.96～42.21，显示了铅源的物质成熟度较高，要求最佳物源是浅变质化学-碎屑沉积建造，恰好与本区泥盆构造层为浅变质细碎屑岩夹薄层灰岩系的特征一致，部分低Sr和Pb同位素比值的成矿物质可能来自于作为围岩的花岗斑岩和/或者基地物质。 总结前人阳山金矿床的H-O-C同位素体系的研究得出，初始成矿流体来源于碳酸盐地层或相似岩石建造的变质或/和改造脱水，成矿流体系统从早到晚、从深到浅，由变质热液演变为大气降水热液。与本文得出的结论一致。 总而言之， 阳山金矿矿成矿流体的来源早期具有变质水特征，应来自赋矿地层或相似岩性组合的改造或变质脱水作用，晚阶段大气水为主的流体性质。成矿物质主要来自于赋矿围岩。流体经过作为部分围岩的花岗斑岩时从中萃取少部分成矿物质，而导致了少部分的低锶、铅同位素的矿石硫化物组成。 在中生代扬子板块北缘（包括碧口地块）向南秦岭陆陆碰撞过程中，扬子北缘板片沿勉略断裂向北俯冲到南秦岭之下，下插板片增温增压，发生变质、脱水和部分熔融。碰撞中期，构造背景由挤压向伸展转变，减压增温的环境导致大量变质流体沿深大断裂向上运移，不断萃取围岩中大量成矿元素，并将成矿元素搬运至有利于流体聚集、成矿物质卸载的空间，使成矿物质富集成矿。阳山金矿床定位于泥盆系构造层中，成矿时代为190Ma左右，紧随花岗斑岩侵入作用（220Ma左右），主成矿作用发生于碰撞作用由挤压-伸展转变期的减压增温环境。成岩、成矿模式与CMF模式吻合。 阳山超大型金矿是世界罕见的碰撞造山带内类卡林型金矿床，其地质地球化学特征复杂、独特，流体性质主要与造山型金矿一致，矿床地质主要与卡林型金矿一致，部分特征兼与造山型和卡林型两类矿床之间，花岗斑岩本身成矿的特点又为阳山所特有，总体具有造山型向卡林型金矿过渡的性质。因此建议以“秦岭式”或“阳山式”类卡林型金矿床代表与阳山金矿具有类似成矿背景及地球化学性质的矿床。

The Hawaiian Islands : their progress and condition under missionary labors / by Rufus Anderson.

http://moa.umdl.umich.edu/cgi/sgml/moa-idx?notisid=ALQ8090

Evaluation of tumor response, disease control, progression-free survival, and time to progression as potential surrogate end points in metastatic breast cancer.

PURPOSE: Overall survival (OS) can be observed only after prolonged follow-up, and any potential effect of first-line therapies on OS may be confounded by the effects of subsequent therapy. We investigated whether tumor response, disease control, progression-free survival (PFS), or time to progression (TTP) could be considered a valid surrogate for OS to assess the benefits of first-line therapies for patients with metastatic breast cancer. PATIENTS AND METHODS: Individual patient data were collected on 3,953 patients in 11 randomized trials that compared an anthracycline (alone or in combination) with a taxane (alone or in combination with an anthracycline). Surrogacy was assessed through the correlation between the end points as well as through the correlation between the treatment effects on the end points. RESULTS: Tumor response (survival odds ratio [OR], 6.2; 95% CI, 5.3 to 7.0) and disease control (survival OR, 5.5; 95% CI, 4.8 to 6.3) were strongly associated with OS. PFS (rank correlation coefficient, 0.688; 95% CI, 0.686 to 0.690) and TTP (rank correlation coefficient, 0.682; 95% CI, 0.680 to 0.684) were moderately associated with OS. Response log ORs were strongly correlated with PFS log hazard ratios (linear coefficient [rho], 0.96; 95% CI, 0.73 to 1.19). Response and disease control log ORs and PFS and TTP log hazard ratios were poorly correlated with log hazard ratios for OS, but the confidence limits of rho were too wide to be informative. CONCLUSION: No end point could be demonstrated as a good surrogate for OS in these trials. Tumor response may be an acceptable surrogate for PFS.

Regulation of G protein-coupled receptor signaling by scaffold proteins.

The actions of many hormones and neurotransmitters are mediated through stimulation of G protein-coupled receptors. A primary mechanism by which these receptors exert effects inside the cell is by association with heterotrimeric G proteins, which can activate a wide variety of cellular enzymes and ion channels. G protein-coupled receptors can also interact with a number of cytoplasmic scaffold proteins, which can link the receptors to various signaling intermediates and intracellular effectors. The multicomponent nature of G protein-coupled receptor signaling pathways makes them ideally suited for regulation by scaffold proteins. This review focuses on several specific examples of G protein-coupled receptor-associated scaffolds and the roles they may play in organizing receptor-initiated signaling pathways in the cardiovascular system and other tissues.

The Ontogeny of Mucosal and Systemic Antibody Responses to HIV-1 Infection

The humoral immune system plays a critical role in the clearance of numerous pathogens. In the setting of HIV-1 infection, the virus infects, integrates its genome into the host's cells, replicates, and establishes a reservoir of virus-infected cells. The initial antibody response to HIV-1 infection is targeted to non-neutralizing epitopes on HIV-1 Env gp41, and when a neutralizing response does develop months after transmission, it is specific for the autologous founder virus and the virus escapes rapidly. After continuous waves of antibody mediated neutralization and viral escape, a small subset of infected individuals eventually develop broad and potent heterologous neutralizing antibodies years after infection. In this dissertation, I have studied the ontogeny of mucosal and systemic antibody responses to HIV-1 infection by means of three distinct aims: 1. Determine the origin of the initial antibody response to HIV-1 infection. 2. Characterize the role of restricted VH and VL gene segment usage in shaping the antibody response to HIV-1 infection. 3. Determine the role of persistence of B cell clonal lineages in shaping the mutation frequencies of HIV-1 reactive antibodies.

After the introduction (Chapter 1) and methods (Chapter 2), Chapter 3 of this dissertation describes a study of the antibody response of terminal ileum B cells to HIV-1 envelope (Env) in early and chronic HIV-1 infection and provides evidence for the role of environmental antigens in shaping the repertoire of B cells that respond to HIV-1 infection. Previous work by Liao et al. demonstrated that the initial plasma cell response in the blood to acute HIV-1 infection is to gp41 and is derived from a polyreactive memory B cell pool. Many of these antibodies cross-reacted with commensal bacteria, Therefore, in Chapter 3, the relationship of intestinal B cell reactivity with commensal bacteria to HIV-1 infection-induced antibody response was probed using single B cell sorting, reverse transcription and nested polymerase chain reaction (RT- PCR) methods, and recombinant antibody technology. The dominant B cell response in the terminal ileum was to HIV-1 envelope (Env) gp41, and 82% of gp41- reactive antibodies cross-reacted with commensal bacteria whole cell lysates. Pyrosequencing of blood B cells revealed HIV-1 antibody clonal lineages shared between ileum and blood. Mutated IgG antibodies cross-reactive with both Env gp41 and commensal bacteria could also be isolated from the terminal ileum of HIV-1 uninfected individuals. Thus, the antibody response to HIV-1 can be shaped by intestinal B cells stimulated by commensal bacteria prior to HIV-1 infection to develop a pre-infection pool of memory B cells cross-reactive with HIV-1 gp41.

Chapter 4 details the study of restricted VH and VL gene segment usage for gp41 and gp120 antibody induction following acute HIV-1 infection; mutations in gp41 lead to virus enhanced neutralization sensitivity. The B cell repertoire of antibodies induced in a HIV-1 infected African individual, CAP206, who developed broadly neutralizing antibodies (bnAbs) directed to the HIV-1 envelope gp41 membrane proximal external region (MPER), is characterized. Understanding the selection of virus mutants by neutralizing antibodies is critical to understanding the role of antibodies in control of HIV-1 replication and prevention from HIV-1 infection. Previously, an MPER neutralizing antibody, CAP206-CH12, with the binding footprint identical to that of MPER broadly neutralizing antibody 4E10, that like 4E10 utilized the VH1-69 and VK3-20 variable gene segments was isolated from this individual (Morris et al., 2011). Using single B cell sorting, RT- PCR methods, and recombinant antibody technology, Chapter 4 describes the isolation of a VH1-69, Vk3-20 glycan-dependent clonal lineage from CAP206, targeted to gp120, that has the property of neutralizing a neutralization sensitive CAP206 transmitted/founder (T/F) and heterologous viruses with mutations at amino acids 680 or 681 in the MPER 4E10/CH12 binding site. These data demonstrate sites within the MPER bnAb epitope (aa 680-681) in which mutations can be selected that lead to viruses with enhanced sensitivity to autologous and heterologous neutralizing antibodies.

In Chapter 5, I have completed a comparison of evolution of B cell clonal lineages in two HIV-1 infected individuals who have a predominant VH1-69 response to HIV-1 infection--one who produces broadly neutralizing MPER-reactive mAbs and one who does not. Autologous neutralization in the plasma takes ~12 weeks to develop (Gray et al., 2007; Tomaras et al., 2008b). Only a small subset of HIV-1 infected individuals develops high plasma levels of broad and potent heterologous neutralization, and when it does occur, it typically takes 3-4 years to develop (Euler et al., 2010; Gray et al., 2007; 2011; Tomaras et al., 2011). The HIV-1 bnAbs that have been isolated to date have a number of unusual characteristics including, autoreactivity and high levels of somatic hypermutations, which are typically tightly regulated by immune control mechanisms (Haynes et al., 2005; 2012b; Kwong and Mascola, 2012; Scheid et al., 2009a). The VH mutation frequencies of bnAbs average ~15% but have been shown to be as high as 32% (reviewed in Mascola and Haynes, 2013; Kwong and Mascola, 2012). The high frequency of somatic hypermutations suggests that the B cell clonal lineages that eventually produce bnAbs undergo high-levels of affinity maturation, implying prolonged germinal center (GC) reactions and high levels of T cell help. To study the duration of HIV-1- reactive B cell clonal persistence, HIV-1 reactive and non HIV-1- reactive B cell clonal lineages were isolated from an HIV-1 infected individual that produces bnAbs, CAP206, and an HIV-1 infected individual who does not produce bnAbs, 004-0. Single B cell sorting, RT-PCR and recombinant antibody technology was used to isolate and produce monoclonal antibodies from multiple time points from each individual. B cell sequences clonally related to mAbs isolated by single cell PCR were identified within pyrosequences of longitudinal samples of these two individuals. Both individuals produced long-lived B cell clones that persisted from 0-232 weeks in CAP206, and 0-238 weeks in 004-0. The average length of persistence of clones containing members isolated from two separate time points was 91.5 weeks both individuals. Examples of the continued evolution of clonal lineages were observed in both the bnAb and non-bnAb individual. These data indicated that the ability to generate persistent and evolving B cell clonal lineages occurs in both bnAb and non-bnAb individuals, suggesting that some alternative host or viral factor is critical for the generation of highly mutated broadly neutralizing antibodies.

Together the studies described in Chapter 3-5 show that multiple factors influence the antibody response to HIV-1 infection. The initial antibody response to HIV-1 Env gp41 can be shaped by a B cell response to intestinal commensal bacteria prior to HIV-1 infection. VH and VL gene segment restriction can impact the B cell response to multiple HIV-1 antigens, and virus escape mutations in the MPER can confer enhanced neutralization sensitivity to autologous and heterologous antibodies. Finally, the ability to generate long-lived HIV-1 clonal lineages in and of itself does not confer on the host the ability to produce bnAbs.