Faculty Bibliography

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.

The V3 region of HIV-1 gp120 is important for virus-coreceptor interaction and highly immunogenic. Although most anti-V3 antibodies neutralize only the sensitive Tier 1 viruses, anti-V3 antibodies effective against the more resistant viruses exist, and a better understanding about these antibodies and their epitopes would be beneficial for the development of novel vaccine immunogens against HIV. The HIV-1 isolate JRFL with its cryptic V3 is resistant to most V3-specific monoclonal antibodies (mAbs). However, the V3 mAb 2424 achieves 100% neutralization against JRFL. 2424 is encoded by IgH V3-53 and IgL V2-28 genes, a pairing rarely used by the other V3 mAbs.. 2424 also has distinct binding and neutralization profiles. Studies of 2424-mediated neutralization of JRFL produced with a mannosidase inhibitor further revealed that its neutralizing activity is unaffected by the glycan composition of the virus envelope. To understand the distinct activity of 2424, we determined the crystal structure of 2424 Fab in complex with a JRFL V3 peptide, and showed that the 2424 epitope is located at the tip of the V3 crown (307IHIGPGRAFY318), dominated by interactions with HisP308, ProP313, and ArgP315. The binding mode of 2424 is similar to that of the well-characterized 447-52D, although 2424 is more side-chain dependent. The 2424 epitope is focused on the very apex of V3, away from nearby glycans, facilitating antibody access. This feature distinguishes the 2424 epitope from the other V3 crown epitopes and indicates that the tip of V3 is a potential site to target and incorporate into HIV vaccine immunogens. IMPORTANCE: HIV/AIDS vaccines are crucial for controlling the HIV epidemics that continue to afflict millions of people worldwide. However, HIV vaccine development has been hampered by significant scientific challenges, one of which is the inability of HIV vaccine candidates evaluated thus far to elicit production of potent and broadly neutralizing antibodies. The V3 loop is one of the few immunogenic targets on the virus envelope glycoprotein that can induce neutralizing antibodies, but in many viruses, parts of V3 are inaccessible for antibody recognition. This study examined a V3-specific monoclonal antibody that can completely neutralize HIV-1 JRFL, a virus isolate resistant to most V3 antibodies. Our data reveal that this antibody recognizes the most distal tip of V3 that is not as occluded as other parts of V3. Hence, the epitope of 2424 is in one of the vulnerable sites on the virus that may be exploited in designing HIV vaccine immunogens.

The first and second variable regions (V1V2) of gp120 play vital roles in the functioning of the HIV-1 envelope (Env). V1V2, which harbors multiple glycans and is highly sequence diverse, is located at the Env apex and stabilizes the trimeric gp120 spike on the virion surface. It shields V3 and the co-receptor binding sites in the pre-fusion state and exposes them upon CD4 binding. Data from the RV144 human HIV-1 vaccine trial suggested that antibody responses targeting V1V2 region inversely correlated with the risk of infection; thus, understanding the antigenic structure of V1V2 can contribute to vaccine design. We have determined a crystal structure of a V1V2-scaffold molecule (V1V2ZM109-1FD6) in complex with 830A, a human monoclonal antibody that recognizes a V1V2 epitope overlaping the integrin-binding motif in V2. The structure revealed that V1V2 assumes a five-stranded beta barrel structure with the region of the integrin-binding site (AAs 179-181) included in a "kink" followed by an extra beta strand. The complete barrel structure naturally presents the glycans on its outer surface and packs into its core conserved hydrophobic residues, including the Ile at position 181 which was highly correlated with vaccine efficacy in RV144. The epitope of monoclonal antibody 830A is discontinuous and composed of three segments: 1) Thr175, Tyr177, Leu179 and Asp180 at the kink overlapping the integrin-binding site; 2) Arg153 and Val154 in V1, and 3) Ile194 at the C-terminus of V2. This study thus provides the atomic details of the immunogenic "V2i epitope". IMPORTANCE: Data from the RV144 Phase III clinical trial suggested that the presence of antibodies to the first and second variable regions (V1V2) of gp120 were associated with the modest protection afforded by the vaccine. V1V2 is a highly variable and immunogenic region of the HIV-1 surface glycoprotein gp120, and structural information about this region and its antigenic landscape will be crucial in the design of an effective HIV-1 vaccine. We have determined a crystal structure of V1V2 in complex with human mAb 830A and showed that mAb 830A recognizes a region overlapping the alpha4beta7 integrin-binding site. We also show that V1V2 forms a 5-stranded beta barrel, an elegant structure allowing for sequence variations in the strand-connecting loops while preserving a conserved core.

The fourth conserved region (C4) in the HIV-1 envelope glycoprotein (Env) gp120 is a structural element that is important for its function, as it binds to both the receptor CD4 and the co-receptor CCR5/CXCR4. It has long been known that this region is highly immunogenic and that it harbors B-cell as well as T-cell epitopes. It is the target of a number of antibodies in animal studies, which are called CD4-blockers. However, the mechanism by which the virus shields itself from such antibody responses is not known. Here, we determined the crystal structure of R53 in complex with its epitope peptide using a novel anti-C4 rabbit monoclonal antibody R53. Our data show that although the epitope of R53 covers a highly conserved sequence (433)AMYAPPI(439), it is not available in the gp120 trimer and in the CD4-bound conformation. Our results suggest a masking mechanism to explain how HIV-1 protects this critical region from the human immune system.