Faculty Bibliography

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.

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.

Elucidating the structural basis of antibody (Ab) gene usage and affinity maturation of vaccine-induced Abs can inform the design of immunogens for inducing desired Ab responses in HIV vaccine development. Analyses of monoclonal Abs (mAbs) encoded by the same immunoglobulin genes in different stages of maturation can help to understand the maturation process. We have analyzed four human anti-V3 mAbs with the same VH1-3*01 and VL3-10*01 gene usage. Two mAbs, TA6 and TA7, were developed from a vaccinee in the HIV vaccine phase I trial DP6-001 with a polyvalent DNA prime - protein boost regimen, and two others, 311-11D and 1334, were developed from HIV-infected patients. The somatic hypermutation (SHM) rates in VH of vaccine-induced mAbs are lower than in chronic HIV infection-induced mAbs, while those in VL are comparable. Crystal structures of the antigen-binding fragments (Fabs) in complex with V3 peptides show that these mAbs bind the V3 epitope with a new cradle-binding mode, and that the V3 β-hairpin lies along the antigen-binding groove, which consists of residues from both heavy and light chains. Residues conserved from the germline sequences form specific binding pockets accommodating conserved structural elements of the V3 crown hairpin, predetermining the Ab gene selection, while somatically mutated residues create additional hydrogen bonds, electrostatic interactions, and van der Waals contacts, correlating with an increased binding affinity. Our data provide a unique example of germline sequences determining the primordial antigen-binding sites and SHMs correlating with affinity maturation of Abs induced by vaccine and natural HIV infection.IMPORTANCE Understanding the structural basis of gene usage and affinity maturation for anti-HIV-1 antibodies may help vaccine design and development. Antibodies targeting the highly immunogenic third variable loop (V3) of HIV-1 gp120 provide a unique opportunity for detailed structural investigations. By comparing the sequences and structures of four anti-V3 mAbs at different stages of affinity maturation but of the same V gene usage, two induced by vaccination and another two by chronic infection, we provide a fine example of how germline sequence determines the essential elements for epitope recognition and how affinity maturation improves the antibody's recognition of its epitope.

The GI tract is preferentially targeted during acute/early HIV-1 infection. Consequent damage to the gut plays a central role in HIV pathogenesis. The basis for preferential targeting of gut tissues is not well defined. Recombinant proteins and synthetic peptides derived from HIV and SIV gp120 bind directly to integrin α4β7, a gut-homing receptor. Using both cell-surface expressed α4β7 and a soluble α4β7 heterodimer we demonstrate that its specific affinity for gp120 is similar to its affinity for MAdCAM (its natural ligand). The gp120 V2 domain preferentially engages extended forms of α4β7 in a cation -sensitive manner and is inhibited by soluble MAdCAM. Thus, V2 mimics MAdCAM in the way that it binds to α4β7, providing HIV a potential mechanism to discriminate between functionally distinct subsets of lymphocytes, including those with gut-homing potential. Furthermore, α4β7 antagonists developed for the treatment of inflammatory bowel diseases, block V2 binding to α4β7. A 15-amino acid V2 -derived peptide is sufficient to mediate binding to α4β7. It includes the canonical LDV/I α4β7 binding site, a cryptic epitope that lies 7-9 amino acids amino terminal to the LDV/I, and residues K169 and I181. These two residues were identified in a sieve analysis of the RV144 vaccine trial as sites of vaccine -mediated immune pressure. HIV and SIV V2 mAbs elicited by both vaccination and infection that recognize this peptide block V2-α4β7 interactions. These mAbs recognize conformations absent from the β- barrel presented in a stabilized HIV SOSIP gp120/41 trimer. The mimicry of MAdCAM-α4β7 interactions by V2 may influence early events in HIV infection, particularly the rapid seeding of gut tissues, and supports the view that HIV replication in gut tissue is a central feature of HIV pathogenesis.

Studies using the European rabbit Oryctolagus cuniculus contributed to elucidating numerous fundamental aspects of antibody structure and diversification mechanisms and continue to be valuable for the development and testing of therapeutic humanized polyclonal and monoclonal antibodies. Additionally, during the last two decades, the use of the European rabbit as an animal model has been increasingly extended to many human diseases. This review documents the continuing wide utility of the rabbit as a reliable disease model for development of therapeutics and vaccines and studies of the cellular and molecular mechanisms underlying many human diseases. Examples include syphilis, tuberculosis, HIV-AIDS, acute hepatic failure and diseases caused by noroviruses, ocular herpes, and papillomaviruses. The use of rabbits for vaccine development studies, which began with Louis Pasteur's rabies vaccine in 1881, continues today with targets that include the potentially blinding HSV-1 virus infection and HIV-AIDS. Additionally, two highly fatal viral diseases, rabbit hemorrhagic disease and myxomatosis, affect the European rabbit and provide unique models to understand co-evolution between a vertebrate host and viral pathogens.