Jovana Golijanin

A single injection of anti-HIV-1 antibodies protects against repeated SHIV challenges

Despite the success of potent anti-retroviral drugs in controlling human immunodeficiency virus type 1 (HIV-1) infection, little progress has been made in generating an effective HIV-1 vaccine. Although passive transfer of anti-HIV-1 broadly neutralizing antibodies can protect mice or macaques against a single high-dose challenge with HIV or simian/human (SIV/HIV) chimaeric viruses (SHIVs) respectively, the long-term efficacy of a passive antibody transfer approach for HIV-1 has not been examined. Here we show, on the basis of the relatively long-term protection conferred by hepatitis A immune globulin, the efficacy of a single injection (20 mg kg−1) of four anti-HIV-1-neutralizing monoclonal antibodies (VRC01, VRC01-LS, 3BNC117, and 10-1074 (refs 9, 10, 11, 12)) in blocking repeated weekly low-dose virus challenges of the clade B SHIVAD8. Compared with control animals, which required two to six challenges (median = 3) for infection, a single broadly neutralizing antibody infusion prevented virus acquisition for up to 23 weekly challenges. This effect depended on antibody potency and half-life. The highest levels of plasma-neutralizing activity and, correspondingly, the longest protection were found in monkeys administered the more potent antibodies 3BNC117 and 10-1074 (median = 13 and 12.5 weeks, respectively). VRC01, which showed lower plasma-neutralizing activity, protected for a shorter time (median = 8 weeks). The introduction of a mutation that extends antibody half-life into the crystallizable fragment (Fc) domain of VRC01 increased median protection from 8 to 14.5 weeks. If administered to populations at high risk of HIV-1 transmission, such an immunoprophylaxis regimen could have a major impact on virus transmission.

Early antibody therapy can induce long-lasting immunity to SHIV

Highly potent and broadly neutralizing anti-HIV-1 antibodies (bNAbs) have been used to prevent and treat lentivirus infections in humanized mice, macaques, and humans. In immunotherapy experiments, administration of bNAbs to chronically infected animals transiently suppresses virus replication, which invariably returns to pre-treatment levels and results in progression to clinical disease. Here we show that early administration of bNAbs in a macaque simian/human immunodeficiency virus (SHIV) model is associated with very low levels of persistent viraemia, which leads to the establishment of T-cell immunity and resultant long-term infection control. Animals challenged with SHIVAD8-EO by mucosal or intravenous routes received a single 2-week course of two potent passively transferred bNAbs (3BNC117 and 10-1074 (refs 13, 14)). Viraemia remained undetectable for 56–177 days, depending on bNAb half-life in vivo. Moreover, in the 13 treated monkeys, plasma virus loads subsequently declined to undetectable levels in 6 controller macaques. Four additional animals maintained their counts of T cells carrying the CD4 antigen (CD4+) and very low levels of viraemia persisted for over 2 years. The frequency of cells carrying replication-competent virus was less than 1 per 106 circulating CD4+ T cells in the six controller macaques. Infusion of a T-cell-depleting anti-CD8β monoclonal antibody to the controller animals led to a specific decline in levels of CD8+ T cells and the rapid reappearance of plasma viraemia. In contrast, macaques treated for 15 weeks with combination anti-retroviral therapy, beginning on day 3 after infection, experienced sustained rebound plasma viraemia when treatment was interrupted. Our results show that passive immunotherapy during acute SHIV infection differs from combination anti-retroviral therapy in that it facilitates the emergence of potent CD8+ T-cell immunity able to durably suppress virus replication.

Coexistence of potent HIV-1 broadly neutralizing antibodies and antibody-sensitive viruses in a viremic controller.

Three new potent neutralizing antibodies neutralize autologous HIV-1 strains and contribute to viral control in an HIV-1 controller. Antibodies can hold HIV-1 at an impasse Neutralizing antibodies put selective pressure on pathogens to mutate and escape from immune detection, which is one of the reasons why HIV-1 infection is difficult to contain. In this issue, Freund et al. studied samples spanning almost a decade from an individual who naturally controls HIV-1 infection without progressing to AIDS. They discovered three potent antibodies coexisting with viral strains that were sensitive to antibody neutralization, indicating that these antibodies may be contributing to viral control. These antibodies were also able to prevent HIV-1 viremia in humanized mice, demonstrating that the antibodies may be beneficial as passive immunotherapy for infected individuals. Some HIV-1–infected patients develop broad and potent HIV-1 neutralizing antibodies (bNAbs) that when passively transferred to mice or macaques can treat or prevent infection. However, bNAbs typically fail to neutralize coexisting autologous viruses due to antibody-mediated selection against sensitive viral strains. We describe an HIV-1 controller expressing HLA-B57*01 and HLA-B27*05 who maintained low viral loads for 30 years after infection and developed broad and potent serologic activity against HIV-1. Neutralization was attributed to three different bNAbs targeting nonoverlapping sites on the HIV-1 envelope trimer (Env). One of the three, BG18, an antibody directed against the glycan-V3 portion of Env, is the most potent member of this class reported to date and, as revealed by crystallography and electron microscopy, recognizes HIV-1 Env in a manner that is distinct from other bNAbs in this class. Single-genome sequencing of HIV-1 from serum samples obtained over a period of 9 years showed a diverse group of circulating viruses, 88.5% (31 of 35) of which remained sensitive to at least one of the temporally coincident autologous bNAbs and the individual’s serum. Thus, bNAb-sensitive strains of HIV-1 coexist with potent neutralizing antibodies that target the virus and may contribute to control in this individual. When administered as a mix, the three bNAbs controlled viremia in HIV-1YU2–infected humanized mice. Our finding suggests that combinations of bNAbs may contribute to control of HIV-1 infection.

The microanatomic segregation of selection by apoptosis in the germinal center

Light- and dark-zone death dynamics Germinal centers (GCs) are areas within lymphoid organs where mature B cells expand and differentiate during normal immune responses. GCs are separated into two anatomic compartments: the dark zone, where B cells divide and undergo somatic hypermutation, and the light zone, where they are selected for affinity-enhancing mutations after interacting with T follicular helper cells. Mayer et al. studied apoptosis reporter mice and found that both GC zones experience very high rates of apoptosis (see the Perspective by Bryant and Hodgkin). However, the underlying mechanisms were distinct and microanatomically segregated. Light-zo ne B cells underwent apoptosis by default unless they were rescued by positive selection. In contrast, apoptotic dark-zone B cells were highly enriched among cells with genes damaged by random antibody-gene mutations. Science, this issue p. eaao2602; see also p. 171 The selection of germinal center B cells by apoptosis is regulated by microanatomically distinct mechanisms. INTRODUCTION Germinal centers (GCs) are transient microanatomic structures that form in lymphoid organs during an immune response. They are the sites of B cell clonal expansion and affinity maturation, a process that leads to the production of high-affinity antibodies. GCs are highly dynamic and contain activated B cells, specialized T follicular helper (TFH) cells, and antigen-trapping follicular dendritic cells. GCs are organized into two functionally distinct compartments: a dark zone (DZ) and a light zone (LZ). The DZ is the site of rapid cell division and random antibody-gene mutation, which is initiated by activation-induced cytidine deaminase (AID). The mutation process leads to the accumulation of a large number of closely related B cells that carry receptors with distinct antigen-binding properties. Once they stop dividing, DZ B cells migrate to the LZ, where their newly generated B cell receptors (BCRs) are tested: GC B cells with relatively higher-affinity receptors capture and process more antigen, leading to positive selection by interaction with TFH cells. The positively selected LZ B cells return to the DZ, where they undergo further cycles of division and mutation. Concomitantly, small numbers of memory B cells and antibody-secreting plasma cells exit the GC. Together, these processes provide the mechanistic basis for affinity maturation, which is essential for effective vaccination and protection from infections. RATIONALE In addition to producing antibody variants, AID expression is also a threat to the genome. AID can produce double-strand breaks that are substrates for chromosome translocations. It can also produce immunoglobulin (Ig) gene missense mutations and deletions or create self-reactive antibodies. These deleterious mutations need to be selected against. Indeed, histologists have long appreciated large numbers of apoptotic nuclei in the specialized tingible body macrophages found in GCs. However, beyond histology, little is known about the exact rate of GC B cell apoptosis and whether it differs in the DZ and LZ of the GC. Moreover, the mechanisms that cause apoptosis, their relative importance in each GC compartment, and their role in GC B cell selection have not been defined. To examine these questions, we created fluorescent apoptosis-indicator mice and used them to enumerate, isolate, and characterize dying cells in the GC. RESULTS We found that apoptosis is prevalent in both the DZ and LZ compartments of GCs throughout the immune response: up to 50% of GC B cells undergo programmed cell death every 6 hours. Single dying GC B cells were isolated, and their antibody genes were cloned, expressed by transient transfection, and tested for antigen binding and other properties. Apoptotic DZ cells were highly enriched for Ig genes damaged by AID, including missense mutations and deletions. By contrast, dying LZ cells primarily expressed intact antibodies with a range of affinities indistinguishable from GC B cells in the live LZ compartment. By experimentally blocking positive selection and by using reporter mice for Myc, a proto-oncogene, as an indicator of positive selection, we found that apoptosis is the default fate for LZ GC B cells that are not actively positively selected. Thus, LZ GC B cells carrying low-affinity BCRs do not preferentially undergo apoptosis. Instead, apoptosis occurs irrespective of BCR-affinity, and LZ B cells carrying high-affinity BCRs are simply more likely to be positively selected. CONCLUSION Apoptosis is a major feature of GC B cell biology and is required to counterbalance the high rate of proliferation and purge B cells that carry deleterious mutations. Although apoptosis occurs in both the DZ and LZ, the underlying mechanisms of apoptosis in each zone are distinct and microanatomically segregated. These insights into GC B biology are relevant for vaccine design, particularly for pathogens that normally evade effective antibody responses. Germinal center B cells expressing an apoptosis indicator. Intravital two-photon microscopy of GC B cells in popliteal lymph nodes of immunized mice (GC B cells, yellow and green; follicular dendritic cell networks, red). The fluorescence resonance energy transfer–based INDIA reporter was used to visualize and purify dying GC B cells. B cells undergo rapid cell division and affinity maturation in anatomically distinct sites in lymphoid organs called germinal centers (GCs). Homeostasis is maintained in part by B cell apoptosis. However, the precise contribution of apoptosis to GC biology and selection is not well defined. We developed apoptosis-indicator mice and used them to visualize, purify, and characterize dying GC B cells. Apoptosis is prevalent in the GC, with up to half of all GC B cells dying every 6 hours. Moreover, programmed cell death is differentially regulated in the light zone and the dark zone: Light-zone B cells die by default if they are not positively selected, whereas dark-zone cells die when their antigen receptors are damaged by activation-induced cytidine deaminase.

Structure of a Natively-glycosylated HIV-1 Env Reveals a New Mode for VH1-2 Antibody Recognition of the CD4 Binding Site Relevant to Vaccine Design

Background: Structural studies of broadly neutralizing antibodies (bNAbs) bound to Env trimers have revealed mechanisms by which bNAbs targeting various epitopes penetrate the glycan shield to either accommodate or include N-glycans in their epitopes. Although accessibility to the conserved host receptor (CD4) binding site (CD4bs) is restricted by surrounding glycans, VRC01-class bNAbs mimic CD4 binding to share a common mode of gp120 binding and glycan accommodation using a VH1-2*02- derived variable heavy (VH) domain. While attractive candidates for immunogen design, features of VRC01-class bNAbs such as a high degree of somatic hypermutation (SHM) and a short (5-residue) light chain (LC) complementarity determining region 3 (CDRL3) (found in only 1% of human LCs) suggest they might be difficult to elicit through vaccination. However, we recently isolated a VH1-2*02-derived CD4bs bNAb, named IOMA, that includes a normal-length (8 residues) CDRL3. Methods: We used X-ray crystallography to solve the first structure of a fully- and natively-glycosylated Env trimer in complex with IOMA, and the V3-loop-directed bNAb 10-1074. Results: Our structure revealed antibody-vulnerable glycan holes and roles of complex-type N-glycans on Env that are relevant to vaccine design, while also demonstrating that IOMA is a new class of CD4-mimetic bNAb that contains features of both VH1-2/VRC01-class and VH1-46/8ANC131-class bNAbs. Conclusions: Analysis of the native glycan shield on HIV-1 Env allows the first full description of the interplay between heterogeneous untrimmed high-mannose and complex-type N-glycans within the CD4bs, V3-loop, and other epitopes on Env. In addition, the structural characterization of IOMA revealed an alternative pathway from VRC01-class bNAbs relevant to vaccine design, which could more readily lead to an effective vaccine response due to higher frequencies of normal-length CDRL3s compared with the rare 5-residue CDRL3s required for VRC01-class bNAbs, and a lower need for SHMs.