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The V1V2 region of the HIV-1 envelope is the target of several broadly neutralizing antibodies (bNAbs). Antibodies to V1V2 elicited in the RV144 clinical trial correlated with a reduced risk of HIV infection, but these antibodies were without broad neutralizing activity. Antibodies targeting V1V2 also correlated with a reduced viral load in immunized macaques challenged with simian immunodeficiency virus (SIV) or simian/human immunodeficiency virus (SHIV). To focus immune responses on V1V2, we engrafted the native, glycosylated V1V2 domain onto five different multimeric scaffold proteins and conducted comparative immunogenicity studies in macaques. Vaccinated macaques developed high titers of plasma and mucosal antibodies that targeted structurally distinct V1V2 epitopes. Plasma antibodies displayed limited neutralizing activity but were functionally active for ADCC and phagocytosis, which was detectable 1-2 years after immunizations ended. This study demonstrates that multivalent, glycosylated V1V2-scaffold protein immunogens focus the antibody response on V1V2 and are differentially effective at inducing polyfunctional antibodies with characteristics associated with protection.

This article was originally published under a CC BY-NC-SA License, but has now been made available under a CC BY 4.0 License.

PURPOSE OF REVIEW/OBJECTIVE:In humans, only one independent immunologic correlate of reduced risk of HIV infection has been identified: a robust antibody (Ab) response to the V1V2 domain of the gp120 envelope (Env) protein. In recent years, the presence and level of V1V2-specific Abs has also been correlated with protection from SIV and SHIV infections. Here, we review the multitude of studies showing the in-vivo protective effects of V1V2 Abs and review their immunologic characteristics and antiviral functions. RECENT FINDINGS/RESULTS:Structural and immunologic studies have defined four epitope families in the V1V2 domain: one epitope family, V2q, which preferentially presents as a quaternary structure of the Env trimer, and another epitope family (V2qt) which requires the quaternary trimeric Env structure; these two epitope types are recognized by two families of monoclonal Abs (mAbs)-V2q-specific and V2qt-specific mAbs-which display broad and potent neutralizing activity. A third epitope family, V2i, is present as a discontinuous conformational structure that overlays the α4β7 integrin binding motif, and a fourth epitope family (V2p) exists on V2 peptides. Antibodies specific for V2i and V2p epitopes display only poor neutralizing activity but effectively mediate other antiviral activities and have been correlated with control of and/or protection from HIV, SIV and SHIV. Notably, V2q and V2qt Abs have not been induced by any vaccines, but V2p and V2i Abs have been readily induced with various vaccines in nonhuman primates and humans. SUMMARY/CONCLUSIONS:The correlation of vaccine-induced V2p and V2i Abs with protection from HIV, SIV and SHIV suggests that these Ab types are extremely important to induce with prophylactic vaccines.

The adenosine A2a receptor agonist CGS21680 has been suggested to act as an anti‑inflammatory agent that protects against cardiopulmonary bypass (CPB)‑induced organ injury. However, the therapeutic effects of CGS21680 for CPB‑induced lung injury have not been comprehensively evaluated. Using a juvenile rat model, the present study was designed to evaluated whether CGS21680 attenuates CPB‑induced lung injury. Our juvenile rat CPB model was established by 60 min CPB with or without CGS21680 pretreatment (100 µg/kg, in the CPB priming solution). Rats in the Sham group only underwent cannulation and heparinization. Serum and pulmonary levels of inflammatory markers and histological features of pulmonary tissues were analyzed. All juvenile rats survived following CPB. Significantly elevated serum levels of tumor necrosis factor‑α (TNF‑α), myeloperoxidase (MPO) and interleukin‑1β (IL‑1β), and decreased glutathione peroxidase (GSH‑PX) levels were observed in the CPB group compared to the Sham group (all P<0.05). TNF‑α, MPO and IL‑1β were significantly decreased, while GSH‑PX was markedly increased in the CGS group when compared to the CPB group. Consistently, pulmonary tissues from rats in the CPB group showed considerable amounts of damaged pneumocytes, severe edema, and increased alveolar macrophages, and significantly higher lung injury scores compared to the controls. Collectively, these changes were all further attenuated by CGS21680. Pretreatment with CGS21680 before CPB attenuated pulmonary injury, which may be related to the anti‑inflammatory effects of CGS21680 downstream of A2a receptor activation.

BACKGROUND:Bringing antibodies from pre-clinical studies to human trials requires humanization, but this process may alter properties that are crucial for efficacy. Since pathological tau protein is primarily intraneuronal in Alzheimer's disease, the most efficacious antibodies should work both intra- and extracellularly. Thus, changes which impact uptake or antibody binding will affect antibody efficacy. METHODS:Initially, we examined four tau mouse monoclonal antibodies with naturally differing charges. We quantified their neuronal uptake, and efficacy in preventing toxicity and pathological seeding induced by human-derived pathological tau. Later, we generated a human chimeric 4E6 (h4E6), an antibody with well documented efficacy in multiple tauopathy models. We compared the uptake and efficacy of unmodified and chimeric antibodies in neuronal and differentiated neuroblastoma cultures. Further, we analyzed tau binding using ELISA assays. FINDINGS/RESULTS:Neuronal uptake of tau antibodies and their efficacy strongly depends on antibody charge. Additionally, their ability to prevent tau toxicity and seeding of tau pathology does not necessarily go together. Particularly, chimerization of 4E6 increased its charge from 6.5 to 9.6, which blocked its uptake into human and mouse cells. Furthermore, h4E6 had altered binding characteristics despite intact binding sites, compared to the mouse antibody. Importantly, these changes in uptake and binding substantially decreased its efficacy in preventing tau toxicity, although under certain conditions it did prevent pathological seeding of tau. CONCLUSIONS:These results indicate that efficacy of chimeric/humanized tau antibodies should be thoroughly characterized prior to clinical trials, which may require further engineering to maintain or improve their therapeutic potential. FUND: National Institutes of Health (NS077239, AG032611, R24OD18340, R24OD018339 and RR027990, Alzheimer's Association (2016-NIRG-397228) and Blas Frangione Foundation.

Targeting tau with immunotherapies is currently the most common approach taken in clinical trials of patients with Alzheimer's disease. The most prominent pathological feature of tau is its hyperphosphorylation, which may cause the protein to aggregate into toxic assemblies that collectively lead to neurodegeneration. Of the phospho-epitopes, the region around Ser³⁹⁶/Ser⁴⁰⁴ has received particular attention for therapeutic targeting because of its prominence and stability in diseased tissue. Herein, we present the antigen-binding fragment (Fab)/epitope complex structures of three different monoclonal antibodies (mAbs) that target the pSer⁴⁰⁴ tau epitope region. Most notably, these structures reveal an antigen conformation similar to a previously described pathogenic tau epitope, pSer⁴²², which was shown to have a β-strand structure that may be linked to the seeding core in tau oligomers. In addition, we have previously reported on the similarly ordered conformation observed in a pSer³⁹⁶ epitope, which is in tandem with pSer⁴⁰⁴. Our data are the first Fab structures of mAbs bound to this epitope region of the tau protein and support the existence of proteopathic tau conformations stabilized by specific phosphorylation events that are viable targets for immune modulation. The atomic coordinates and structure factors have been deposited in the RCSB Protein Data Bank under accession codes 6DC7 (8B2 apo), 6DC8 (8B2), 6DC9 (h4E6), and 6DCA (6B2).

BACKGROUND:HIV infection is enhanced by cell adhesions that form between infected and uninfected T cells called virological synapses (VS). VS are initiated by an interaction between Env and CD4 on cell surfaces and result in the recruitment of virus assembly to the site of cell-cell contact. However, the recruitment of Env to the VS and its relationship to Gag recruitment is not well defined. RESULTS:To study the trafficking of HIV-1 Env through the VS, we constructed a molecular clone of HIV carrying a green fluorescent protein-Env fusion protein called, HIV Env-isfGFP-∆V1V2. The Env-isfGFP-∆V1V2 fusion protein does not produce virus particles on its own, but can be rescued by cotransfection with full-length HIV constructs and produce virus particles that package the fluorescent Env. These rescued fluorescent Env can participate in VS formation and can be used to directly image CD4-dependent Env transfer across VS from donor to target cells. The movements of fluorescently tagged Gag and Env to the VS and transfer into target cells can be also tracked through live imaging. Time lapse live imaging reveals evidence of limited Env accumulation at the site of cell-cell contact shortly after cell adhesion, followed by Gag re-distribution to contact area. Both Gag and Env can be recruited to form button-like spots characteristic of VS. CONCLUSIONS:Env and Gag are recruited to the VS in a coordinated temporal sequence and subsequently transfer together across the synapse into the target cell. Env accumulations, when observed, are earlier than Gag re-distribution to the contact area during formation of VS.

The results of the RV144 vaccine clinical trial showed a correlation between high level of anti-V1V2 antibodies (Abs) and a decreased risk of acquiring HIV-1 infection. This turned the focus of HIV vaccine design to the induction of elevated levels of anti-V2 Abs to increase vaccine efficacy. In plasma samples from HIV-1 infected Cameroonian individuals, we observed broad variations in levels of anti-V2 Abs, and 6 of the 79 plasma samples tested longitudinally displayed substantial deficiency of V2 Abs. Sequence analysis of the V2 region from plasma viruses and multivariate analyses of V2 characteristics showed a significant difference in several features between V2-deficient and V2-reactive plasma Abs. These results suggest that HIV vaccine immunogens containing a shorter V2 region with fewer glycosylation sites and higher electrostatic charges can be beneficial for induction of a higher level of anti-V2 Abs and thus contribute to HIV vaccine efficacy.

Genetic and immunologic analyses of epidemiologically-linked HIV transmission enable insights into the impact of immune responses on clinical outcomes. Human vaccine trials and animal studies of HIV-1 infection have suggested immune correlates of protection; however, their role in natural infection in terms of protection from disease progression is mostly unknown. Four HIV-1⁺ Cameroonian individuals, three of them epidemiologically-linked in a polygamous heterosexual relationship and one incidence-matched case, were studied over 15 years for heterologous and cross-neutralizing antibody responses, antibody binding, IgA/IgG levels, antibody-dependent cellular cytotoxicity (ADCC) against cells expressing wild-type or CD4-bound Env, viral evolution, Env epitopes, and host factors including HLA-I alleles. Despite viral infection with related strains, the members of the transmission cluster experienced contrasting clinical outcomes including cases of rapid progression and long-term non-progression in the absence of strongly protective HLA-I or CCR5Δ32 alleles. Slower progression and higher CD4/CD8 ratios were associated with enhanced IgG antibody binding to native Env and stronger V1V2 antibody binding responses in the presence of viruses with residue K169 in V2. ADCC against cells expressing Env in the CD4-bound conformation in combination with low Env-specific IgA/IgG ratios correlated with better clinical outcome. This data set highlights for the first time that V1V2-directed antibody responses and ADCC against cells expressing open, CD4-exposed Env, in the presence of low plasma IgA/IgG ratios, can correlate with clinical outcome in natural infection. These parameters are comparable to the major correlates of protection, identified post-hoc in the RV144 vaccine trial; thus, they may also modulate the rate of clinical progression once infected. The findings illustrate the potential of immune correlate analysis in natural infection to guide vaccine development.

447-52D (447) is a human monoclonal antibody that recognizes a conserved epitope in the crown region of the third variable loop (V3) of HIV-1 gp120, and like many anti-HIV-1 antibodies with broad neutralization capabilities, it has a long heavy-chain complementarity determining region (CDRH3). Here, we use a combination of computational mutagenesis and modeling in tandem with fluorescence polarization assays to interrogate the molecular basis of 447 CDRH3 length and the individual contribution of selected CDRH3 residues to affinity. We observe that 447 CDRH3 length provides a large binding surface area and the best enthalpic contributions derived from hydrophobic packing, main-chain hydrogen bonds, electrostatic and van der Waals interactions. We also found out that CDRH3 residue Try100I is critical to 447 binding affinity.