Faculty Bibliography

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.

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.