Faculty Bibliography

The V1V2 region of HIV-1 gp120 harbors a major vulnerable site targeted by a group of broadly neutralizing mAbs such as PG9 through strand-strand recognition. However, this epitope region is structurally polymorphic, as it can also form a helical conformation recognized by RV144 vaccine-induced mAb CH58. This structural polymorphism is a potential mechanism for masking the V1V2 vulnerable site. Designing immunogens that can induce conformation-specific Ab responses may lead to vaccines targeting this vulnerable site. We designed a panel of immunogens engrafting the V1V2 domain into trimeric and pentameric scaffolds in structurally constrained conformations. We also fused V1V2 to an Fc fragment to mimic the unconstrained V1V2 conformation. We tested these V1V2-scaffold proteins for immunogenicity in rabbits and assessed the responses by ELISA and competition assays. Our V1V2 immunogens induced distinct conformation-specific Ab responses. Abs induced by structurally unconstrained immunogens reacted preferentially with V1V2-unconstrained antigens, suggesting recognition of the helical configuration, while Abs induced by the structurally constrained immunogens reacted preferentially with V1V2-constrained antigens, suggesting recognition of the beta-strand conformation. The Ab responses induced by the structurally constrained immunogens were more broadly reactive and had higher titers than those induced by the structurally unconstrained immunogens. Our results demonstrate that immunogens presenting the different structural conformations of the gp120 V1V2 vulnerable site can be designed, and that these immunogens induce distinct Ab responses with epitope conformation specificity. Therefore, these structurally constrained V1V2 immunogens are vaccine prototypes targeting the V1V2 domain of the HIV-1 envelope. IMPORTANCE: Correlates analysis of the RV144 HIV-1 vaccine trial suggested that the presence of antibodies to the V1V2 region of HIV-1 gp120 was responsible for the modest protection observed in the trial. In addition V1V2 harbors one of the key vulnerable sites of HIV-1 Env recognized by a family of broadly neutralizing mAbs such as PG9. Thus V1V2 is a key target for vaccine development. However, this vulnerable site is structurally polymorphic, and designing immunogens that present different conformations is crucial for targeting this site. We show here that such immunogens can be designed and they induced conformation-specific antibody responses in rabbits. Our immunogens are therefore prototypes of vaccine candidates targeting the V1V2 region of HIV-1 Env.

Strong antibody (Ab) responses against V1V2 epitopes of the HIV-1 gp120 envelope (Env) correlated with reduced infection rates in studies of HIV, SHIV, and SIV. In order to focus the Ab response on V1V2, we used six V1V2 sequences and nine scaffold proteins to construct immunogens which were tested using various immunization regimens for their ability to induce cross-reactive and biologically active V2 Abs in rabbits. A prime/boost immunization strategy was employed using gp120 DNA and various V1V2-scaffold proteins. The rabbit polyclonal Ab responses (a) were successfully focused on the V1V2 region, with weak or only transient responses to other Env epitopes, (b) displayed broad cross-reactive binding activity with gp120s and the V1V2 regions of diverse strains from clades B, C, and E, (c) included V2 Abs with specificities similar to those found in HIV-infected individuals, and (d) were detectable >/=1 year after the last boosting dose. Importantly, sera from rabbits receiving V1V2-scaffold immunogens displayed Ab-dependent cellular phagocytosis whereas sera from rabbits receiving only gp120 did not. The results represent the first fully successful example of reverse vaccinology in the HIV vaccine field with rationally-designed epitope-scaffold immunogens inducing Abs that recapitulate the epitope specificity and biologic activity of the human monoclonal Abs from which the immunogens were designed. Moreover, this is the first immunogenicity study using epitope-targeting rationally designed vaccine constructs that induced an Fc-mediated activity associated with protection from infection with HIV, SIV and SHIV. IMPORTANCE: Novel immunogens were designed to focus the antibody response of rabbits on the V1V2 epitopes of HIV-1 gp120 since such antibodies were associated with reduced infection rates of HIV, SIV, and SHIV. The vaccine-induced antibodies were broadly cross-reactive with the V1V2 regions of HIV subtypes B, C and E, and, importantly, facilitated Fc-mediated phagocytosis, an activity not induced upon immunization of rabbits with gp120. This is the first immunogenicity study of vaccine constructs that focuses the antibody response on V1V2 and induces V2-specific antibodies with the ability to mediate phagocytosis, an activity that has been associated with protection from infection with HIV, SIV and SHIV.

The isolation of HIV-1 broadly neutralizing antibodies (bnAbs) has demonstrated the ability of the human immune system to mount effective antibody responses against the virus. To harness this immune potential to elicit similar antibody responses by vaccination, it is important to understand the immunological processes that produce them. Here we review recent advances in crystal structural determinations of HIV-1 bnAb epitopes that directly portray the antigenic landscape of the HIV-1 envelope glycoprotein. We also summarize new immunological concepts implicated in bnAb sequences and their lineage studies.

Proteus mirabilis contributes to a significant number of catheter-associated urinary tract infections, where coordinated regulation of adherence and motility is critical for ascending disease progression. Previously, the mannose-resistant Proteus-like (MR/P) fimbria-associated transcriptional regulator MrpJ has been shown to both repress motility and directly induce the transcription of its own operon, in addition to affecting the expression of a wide range of cellular processes. Interestingly, 14 additional mrpJ paralogs are encoded by the P. mirabilis genome. Looking at a selection of MrpJ paralogs, we discovered that these proteins, which consistently repress motility, also have non-identical functions that include cross-regulation of fimbrial operons. A subset of paralogs, including AtfJ (encoded by the ambient temperature fimbrial operon), Fim8J, and MrpJ, is capable of auto-induction. We identified an element of the atf promoter extending from 487 to 655 nucleotides upstream of the transcriptional start site that is responsive to AtfJ, and we found that AtfJ directly binds this fragment. Mutational analysis of AtfJ revealed that its two identified functions, autoregulation and motility repression, are not invariably linked. Residues within the DNA-binding helix-turn-helix domain are required for motility repression but not necessarily autoregulation. Likewise, the C-terminal domain is dispensable for motility repression but is essential for autoregulation. Supported by a 3D-structural model, we hypothesize the C-terminal domain confers unique regulatory capacities to the AtfJ family of regulators. IMPORTANCE: Balancing adherence with motility is essential for uropathogens to successfully establish a foothold in their host. Proteus mirabilis uses a fimbria-associated transcriptional regulator to switch between these antagonistic processes by increasing fimbrial adherence while simultaneously downregulating flagella. The discovery of multiple related proteins, many of which also function as motility repressors, in the P. mirabilis genome, has raised considerable interest as to their functionality and potential redundancy in this organism. This study provides an important advance by elucidating the non-identical effects of these paralogs on a molecular level. Our mechanistic studies of one member of this group, AtfJ, shed light on how these differing functions may be conferred despite the limited sequence variety exhibited by the paralogous proteins.

Uroplakins (UPs) are major differentiation products of urothelial umbrella cells, playing important roles in forming the permeability barrier, and in the expansion/stabilization of the apical membrane. Further, UPIa serves as a uropathogenic E. coli receptor. While it is understood that UPs are delivered to the apical membrane via fusiform vesicles (FVs), the mechanisms that regulate this exocytic pathway remain poorly understood. Immuno-microscopy of normal and mutant mouse urothelia showed that the UP-delivering FVs contained Rab8/11 and Rab27b/Slac2-a, which mediate apical transport along actin filaments. Subsequently, a Rab27b/Slp2-a complex mediated FV-membrane anchorage before SNARE-mediated and MAL-facilitated apical fusion. We also showed that keratin 20 (K20), which formed a chicken-wire network 150-300 nm below the apical membrane and had hole sizes allowing FV passage, defined a subapical compartment containing FVs primed and strategically located for fusion. Finally, we showed that Rab8/11 and Rab27b function in the same pathway, that Rab27b-knockout leads to uroplakin and Slp2-a destabilization, and that Rab27b works upstream from MAL. These data support a unifying model in which UP cargoes are targeted for apical insertion via sequential interactions with Rabs and their effectors, SNAREs and MAL, and in which K20 plays a key role in regulating vesicular trafficking.

Antibodies (Abs) specific for the V3 loop of the HIV-1 gp120 envelope neutralize most Tier 1 and many Tier 2 viruses and are present in essentially all HIV-infected individuals as well as immunized humans and animals. Vaccine-induced V3 Abs are associated with reduced HIV infection rates in humans, and affected the nature of transmitted viruses in infected vaccinees despite the fact that V3 is often occluded in the envelope trimer. Here, we link structural and experimental data showing how conformational alterations of the envelope trimer render viruses exceptionally sensitive to V3 Abs. The experiments interrogated the neutralization sensitivity of pseudoviruses with single amino acid mutations in various regions of gp120 that were predicted to alter packing of the V3 loop in the Env trimer. The results indicate that the V3 loop is meta-stable in the envelope trimer on the virion surface, flickering between states in which V3 is either occluded or available for binding to chemokine receptors (leading to infection) and to V3 Abs (leading to virus neutralization). The "spring loaded" V3 in the envelope trimer is easily "released" by disruption of the stability of the "V3 pocket" in the unliganded trimer or disruption of favorable V3/pocket interactions. Formation of the V3 pocket requires appropriate positioning of the V1V2 domain, which is in turn dependent on the conformation of the bridging sheet, and on the stability of the V1V2 B-C strand-connecting loop. IMPORTANCE: Antibodies levels to the third variable regions (V3) of the HIV envelope protein correlate with reduced HIV infection rates. Previous studies showed that V3 is often occluded as it sits in a pocket of the envelope trimer on the surface of virions, however, the trimer is flexible, allowing occluded portions of the envelope (like V3) to flicker into an exposed position that binds antibodies. Here we provide a systematic interrogation of mechanisms by which single amino acid changes in various regions of gp120: (a) render viruses sensitive to neutralization by V3 antibodies, (b) result in altered packing of the V3 loop, and (c) activate an "open" conformation that exposes V3 to the effects of V3 Abs. Taken together, these and previous studies explain how V3 antibodies can protect against HIV-1 infection, and why they should be one of the targets of vaccine-induced antibodies.

During urinary tract infection (UTI), the second most common bacterial infection, dynamic interactions take place between uropathogenic E. coli (UPEC) and host urothelial cells. While significant strides have been made in the identification of the virulence factors of UPEC, our understanding of how the urothelial cells mobilize innate defenses against the invading UPEC remains rudimentary. Here we show that mouse urothelium responds to the adhesion of type 1-fimbriated UPEC by rapidly activating the canonical NF-kappaB selectively in terminally differentiated, superficial (umbrella) cells. This activation depends on a dual ligand/receptor system, one between FimH adhesin and uroplakin Ia and another between lipopolysaccharide and Toll-like receptor 4. When activated, all the nuclei (up to 11) of a multinucleated umbrella cell are affected, leading to significant amplification of proinflammatory signals. Intermediate and basal cells of the urothelium undergo NF-kappaB activation only if the umbrella cells are detached or if the UPEC persistently express type 1-fimbriae. Inhibition of NF-kappaB prevents the urothelium from clearing the intracellular bacterial communities, leading to prolonged bladder colonization by UPEC. Based on these data, we propose a model of dual ligand/receptor system in innate urothelial defenses against UPEC.