Faculty Bibliography

Current serological assays that are used for cross-sectional HIV incidence estimation have been shown to misclassify individuals with chronic infection. Limited information exists on the performance of cross-sectional incidence assays in Central Africa. HIV-positive individuals from Cameroon who were infected for at least one year or two years were evaluated to determine the false recent ratio of a two-assay algorithm which includes the Limiting Antigen Avidity (LAg-Avidity) assay (ODn<1.5) and HIV viral load (>1000 copies/mL). The subject-level false recent ratio was 5.3% (95% CI, 2.1-10.5) for individuals infected for >/=1 year and 3.9% (95% CI, 0.8-11.0) for individuals infected for >/= 2 years. These data suggest that the LAg-Avidity plus viral load incidence algorithm may overestimate HIV incidence rates in Central Africa.

The global intensification of ART can lead to increased rates of HIV drug resistance (HIVDR) mutations in treated, and also in ART naive patients. ART naive HIV-1 infected patients from Cameroon were subjected to a multimethod HIVDR analysis using Amplification Refractory Mutation System (ARMS)-PCR, Sanger sequencing, and longitudinal Next-Generation Sequencing (NGS), to determine their mutation profiles for K103N, Y181C, K65R, M184V, and T215F/Y. We processed 66 ART-naive HIV-1+ patients with highly diverse subtypes, underlining the predominance of CRF02_AG and the increasing rate of F2 and other recombinant forms in Cameroon. We compared three resistance testing methods for 5 major mutation sites. Using Sanger sequencing, the overall prevalence of HIVDR mutations was 7.6% (5/66) and included all studied mutations except K65R. Comparing ARMS-PCR with Sanger sequencing as a reference, we obtained a sensitivity of 100% (5/5) and a specificity of 95% (58/61), caused by three false positive calls with ARMS-PCR. For 32/66 samples we obtained NGS data and we observed two additional mismatches made up by minority variants (7%, 18%) that might not be clinically relevant. Longitudinal NGS analyses revealed changes in HIVDR mutations in all five positive subjects that could not be attributed to treatment. In one of these cases, superinfection led to the temporary masking of a resistant virus.HIVDR mutations can be sensitively detected by ARMS-PCR and Sequencing methods with comparable performance. Longitudinal changes in HIVDR mutations have to be considered even in the absence of treatment.

OBJECTIVE: To investigate the viral genetic evolution, spatial origins and patterns of transmission of HIV-1 subtype B in Shanghai, China. METHODS: A total of 242 Shanghai subtype B and 1519 reference pol sequences were subjected to phylogenetic inference and genetic transmission network analyses. RESULTS: Phylogenetic analysis revealed that subtype B strains circulating in Shanghai were genetically diverse and closely associated with viral sequence lineages in Beijing (76 of 242 [31.4%]), Central China (Henan/Hebei/Hunan/Hubei) (43 of 242 [17.8%]), Chinese Taiwan (20 of 242 [8.3%]), Japan (6 of 242 [2.5%]), and Korea (7 of 242 [2.9%]), suggesting multiple introductions into Shanghai from mainland China and Taiwan, Japan, and Korea. Interestingly, a monophyletic Shanghai lineage (SH-L) (36 of 242 [14.9%]) of HIV-1 subtype B most likely originated from an Argentine strain, transferred through Liaoning infected individuals. In-depth analyses of 195 Shanghai subtype B sequences revealed that a total of 37.9% (n = 74) sequences contributed to 35 transmission networks, whereof 33.8% (n = 25) of the sequences associated with infected individuals from other provinces. CONCLUSIONS: Our new findings reflect the evolution complexity and transmission dynamics of HIV-1 subtype B in Shanghai, which would provide critical information for the design of effective prevention measures against HIV transmission.

HIV superinfection describes the sequential infection of an individual with two or more unrelated HIV strains. Intersubtype superinfection has been shown to cause a broader and more potent heterologous neutralizing antibody response when compared to singly infected controls, yet the effects of intrasubtype superinfection remain controversial. Longitudinal samples were analyzed phylogenetically for pol and env regions using Next-Generation Sequencing and envelope cloning. The impact of CRF02_AG intrasubtype superinfection was assessed for heterologous neutralization and antibody binding responses. We compared two cases of CRF02_AG intrasubtype superinfection that revealed complete replacement of the initial virus by superinfecting CRF02_AG variants with signs of recombination. NYU6564, who became superinfected at an early time point, exhibited greater changes in antibody binding profiles and generated a more potent neutralizing antibody response post-superinfection compared to NYU6501. In contrast, superinfection occurred at a later time point in NYU6501 with strains harboring significantly longer V1V2 regions with no observable changes in neutralization patterns. Here we show that CRF02_AG intrasubtype superinfection can induce a cross-subtype neutralizing antibody response, and our data suggest timing and/or superinfecting viral envelope characteristics as contributing factors. These results highlight differential outcomes in intrasubtype superinfection and provide the first insight into cases with CRF02_AG, the fourth most prevalent HIV-1 strain worldwide.

BACKGROUND: HIV-1 is decorated with trimeric glycoprotein spikes that enable infection by engaging CD4 and a chemokine coreceptor, either CCR5 or CXCR4. The variable loop 3 (V3) of the HIV-1 envelope protein (Env) is the main determinant for coreceptor usage. The predominant CCR5 using (R5) HIV-1 Env has been intensively studied in function and structure, whereas the trimeric architecture of the less frequent, but more cytopathic CXCR4 using (X4) HIV-1 Env is largely unknown, as are the consequences of sequence changes in and near V3 on antigenicity and trimeric Env structure. RESULTS: Soluble trimeric gp140 Env constructs were used as immunogenic mimics of the native spikes to analyze their antigenic properties in the context of their overall 3D structure. We generated soluble, uncleaved, gp140 trimers from a prototypic T-cell line-adapted (TCLA) X4 HIV-1 strain (NL4-3) and a hybrid (NL4-3/ADA), in which the V3 spanning region was substituted with that from the primary R5 isolate ADA. Compared to an ADA (R5) gp140, the NL4-3 (X4) construct revealed an overall higher antibody accessibility, which was most pronounced for the CD4 binding site (CD4bs), but also observed for mAbs against CD4 induced (CD4i) epitopes and gp41 mAbs. V3 mAbs showed significant binding differences to the three constructs, which were refined by SPR analysis. Of interest, the NL4-3/ADA construct with the hybrid NL4-3/ADA CD4bs showed impaired CD4 and CD4bs mAb reactivity despite the presence of the essential elements of the CD4bs epitope. We obtained 3D reconstructions of the NL4-3 and the NL4-3/ADA gp140 trimers via electron microscopy and single particle analysis, which indicates that both constructs inherit a propeller-like architecture. The first 3D reconstruction of an Env construct from an X4 TCLA HIV-1 strain reveals an open conformation, in contrast to recently published more closed structures from R5 Env. Exchanging the X4 V3 spanning region for that of R5 ADA did not alter the open Env architecture as deduced from its very similar 3D reconstruction. CONCLUSIONS: 3D EM analysis showed an apparent open trimer configuration of X4 NL4-3 gp140 that is not modified by exchanging the V3 spanning region for R5 ADA.

Peptides are ideally suited to mimic natural ligands and thereby function in an antagonistic or agonistic way. Furthermore they are able to physiologically disrupt functional complexes due to their small size and specific binding properties. Proteins form homo- or heteromeric (macro)molecular complexes and intricate networks by interacting with small molecules, peptides, nucleic acids or other proteins. On average, five interaction partners have been estimated for any given cellular protein, illustrating the complexity of the formed 'interactomes' and the impact of their investigation. Many protein-protein interactions are mediated by hot-spots, which comprise only a small part of the large binding interface but account for 80% of the binding energy. Thus, these hot spots provide an 'Achilles heel' for pharmaceutical interventions aiming at the disruption of functional protein-protein complexes. Methods to select peptides for defined target structures include display technologies on phages, ribosomes or yeast, and the yeast-two-hybrid system. Once selected, these peptides can be optimized for their binding affinity using peptide arrays. Stabilization of biologically unstable peptides is achieved by the introduction of non-natural amino acids to form so-called peptidomimetics that are resistant to cellular proteases. Moreover, lipocalins and peptide aptamers represent scaffolded binding structures with unique binding characteristics and enhanced stability. In case of extracellular targets, like cell surface receptors or pathogens in patients` plasma, peptide inhibitors have direct access. Addressing intracellular targets with peptides is more difficult since short hydrophilic peptides generally do not cross plasma membranes on their own. However, intracellular uptake of peptides can be achieved by coupling to carrier systems like liposomes or nanoparticles or upon fusion to a protein transduction domain. Alternatively, peptides may be expressed within cells after transduction with viral vectors in a gene therapy setting. This review will summarize the broad potential of peptides as drugs, with special emphasis on peptides which inhibit protein-protein interactions.