Analysis of correlates of risk of infection in the RV144 HIV-1 vaccine efficacy trial demonstrated that plasma IgG against the HIV-1 envelope (Env) variable region 1 and 2 inversely correlated with risk, whereas HIV-1 Env-specific plasma IgA responses directly correlated with risk. In the secondary analysis, antibody-dependent cellular cytotoxicity (ADCC) was another inverse correlate of risk, but only in the presence of low plasma IgA Env-specific antibodies. Thus, we investigated the hypothesis that IgA could attenuate the protective effect of IgG responses through competition for the same Env binding sites. We report that Env-specific plasma IgA/IgG ratios are higher in infected than in uninfected vaccine recipients in RV144. Moreover, Env-specific IgA antibodies from RV144 vaccinees blocked the binding of ADCC-mediating mAb to HIV-1 Env glycoprotein 120 (gp120). An Env-specific monomeric IgA mAb isolated from an RV144 vaccinee also inhibited the ability of natural killer cells to kill HIV-1-infected CD4 + T cells coated with RV144-induced IgG antibodies. We show that monomeric Env-specific IgA, as part of postvaccination polyclonal antibody response, may modulate vaccine-induced immunity by diminishing ADCC effector function.T he phase III RV144 ALVAC/AIDSVAX B/E HIV-1 vaccine efficacy trial in Thailand demonstrated 31.2% estimated vaccine efficacy through 42 mo of follow-up (1). Analysis of correlates of risk of infection indicated that envelope (Env)-specific plasma antibody responses were associated with a lower infection risk in vaccinees (2). Though plasma Env variable region 1 and 2 (V1/V2) IgG correlated with decreased infection risk, high levels of anti-HIV-1 Env plasma IgA correlated with increased infection risk (2). Interaction analyses demonstrated that, in the presence of low IgA Env antibodies, antibody-dependent cellular cytotoxicity (ADCC) responses inversely correlated with risk of infection, whereas in the presence of high IgA Env plasma antibodies, there was no correlation with risk of infection (2). Because there was no overall enhancement of infection risk in the trial (1), we hypothesized that Env IgA might block potentially protective effector functions of Env IgG antibodies.Antibody function depends, in part, on ability to bind to Fc receptors (FcR) on effector cells. The antibody isotype and subclass influences its affinity for different cellular FcRs (3, 4). IgG antibodies that mediate ADCC through natural killer (NK) cells bind to FcγRIIIa (CD16). In contrast, IgA antibodies do not bind to FcγRIIIa, but, rather, have high affinity for FcαRI (CD89) expressed by monocytes/macrophages and polymorphonuclear cells (PMN). This differential profile of FcR binding by IgG and IgA antibodies impacts the effector function capabilities of these antibody isotypes.Here, we examined plasma IgA and monomeric IgA monoclonal antibodies from RV144 vaccine recipients to test the hypothesis that some fraction of the vaccine-elicited IgA response could block IgG-mediated ADCC function. We found that a f...

The 18-kb Xist long noncoding RNA (lncRNA) is essential for X-chromosome inactivation during female eutherian mammalian development. Global structural architecture, cell-induced conformational changes, and protein-RNA interactions within Xist are poorly understood. We used selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) to examine these features of Xist at single-nucleotide resolution both in living cells and ex vivo. The Xist RNA forms complex welldefined secondary structure domains and the cellular environment strongly modulates the RNA structure, via motifs spanning onehalf of all Xist nucleotides. The Xist RNA structure modulates protein interactions in cells via multiple mechanisms. For example, repeatcontaining elements adopt accessible and dynamic structures that function as landing pads for protein cofactors. Structured RNA motifs create interaction domains for specific proteins and also sequester other motifs, such that only a subset of potential binding sites forms stable interactions. This work creates a broad quantitative framework for understanding structure-function interrelationships for Xist and other lncRNAs in cells.ong noncoding RNAs (lncRNAs) play central roles in the regulation of gene expression through interactions with numerous protein partners (1) and are necessary for normal health and development (2, 3). The 18-kb Xist lncRNA is essential for X-chromosome inactivation during female eutherian mammalian development and is an archetype of gene-silencing lncRNAs. During the early stages of X inactivation, Xist accumulates in cis around the future inactive X chromosome and recruits protein complexes that apply repressive chromatin modifications, leading to stable gene silencing (3,4).Genetic deletion studies have demarcated several broad regions of function within Xist. Several tandem repeat regions (labeled A-F in the mouse) show moderate conservation (5-7), and at least two of these, repeat A and the rodent-specific repeat C, are implicated in silencing and localization to the inactive X. Deletion of the final 7.5-kb exon of Xist causes a defect in its localization (8), and the 1.5-kb region encompassing repeats F and B is required for accumulation of heterochromatic marks over the inactive X (4); however, beyond these initial characterizations, the mechanisms by which gene silencing, heterochromatinization, and localization of Xist on the X chromosome occur are not well understood. In particular, the role of RNA structure in orchestrating these distinct functions remains unclear.Several previous studies have suggested the importance of RNA structures in specific regions of Xist (9-12), but overall, the locations and structures of functional domains within Xist are poorly defined. Detailed structural maps of other functional RNAs, such as ribosomal RNAs (13) and the HIV RNA genome (14-16), have been fundamental to understanding the mechanisms by which individual domains within large RNAs execute discrete cellular functions. A detailed an...

There are no available vaccines for dengue, the most important mosquito-transmitted viral disease. Mechanistic studies with anti-dengue virus (DENV) human monoclonal antibodies (hMAbs) provide a rational approach to identify and characterize neutralizing epitopes on DENV structural proteins that can serve to inform vaccine strategies. Here, we report a class of hMAbs that is likely to be an important determinant in the human humoral response to DENV infection. In this study, we identified and characterized three broadly neutralizing anti-DENV hMAbs: 4.8A, D11C, and 1.6D. These antibodies were isolated from three different convalescent patients with distinct histories of DENV infection yet demonstrated remarkable similarities. All three hMAbs recognized the E glycoprotein with high affinity, neutralized all four serotypes of DENV, and mediated antibody-dependent enhancement of infection in Fc receptor-bearing cells at subneutralizing concentrations. The neutralization activities of these hMAbs correlated with a strong inhibition of virus-liposome and intracellular fusion, not virus-cell binding. We mapped epitopes of these antibodies to the highly conserved fusion loop region of E domain II. Mutations at fusion loop residues W101, L107, and/or G109 significantly reduced the binding of the hMAbs to E protein. The results show that hMAbs directed against the highly conserved E protein fusion loop block viral entry downstream of virus-cell binding by inhibiting E protein-mediated fusion. Characterization of hMAbs targeting this region may provide new insights into DENV vaccine and therapeutic strategies.

For decades, human infections with Zika virus (ZIKV), a mosquito-transmitted flavivirus, were sporadic, associated with mild disease, and went underreported since symptoms were similar to other acute febrile diseases. Recent reports of severe disease associated with ZIKV have greatly heightened awareness. It is anticipated that ZIKV will continue to spread in the Americas and globally where competent Aedes mosquito vectors are found. Dengue virus (DENV), the most common mosquito-transmitted human flavivirus, is both well-established and the source of outbreaks in areas of recent ZIKV introduction. DENV and ZIKV are closely related, resulting in substantial antigenic overlap. Through antibody-dependent enhancement (ADE), anti-DENV antibodies can enhance the infectivity of DENV for certain classes of immune cells, causing increased viral production that correlates with severe disease outcomes. Similarly, ZIKV has been shown to undergo ADE in response to antibodies generated by other flaviviruses. We tested the neutralizing and enhancing potential of well-characterized broadly neutralizing human anti-DENV monoclonal antibodies (HMAbs) and human DENV immune sera against ZIKV using neutralization and ADE assays. We show that anti-DENV HMAbs, cross-react, do not neutralize, and greatly enhance ZIKV infection in vitro. DENV immune sera had varying degrees of neutralization against ZIKV and similarly enhanced ZIKV infection. Our results suggest that pre-existing DENV immunity may enhance ZIKV infection in vivo and may lead to increased disease severity. Understanding the interplay between ZIKV and DENV will be critical in informing public health responses and will be particularly valuable for ZIKV and DENV vaccine design and implementation strategies.