Neutralising properties of peptides derived from CXCR4 extracellular loops towards CXCL12 binding and HIV-1 infection.

The chemokine receptor CXCR4 interacts with a single endogenous chemokine, CXCL12, and regulates a wide variety of physiological and pathological processes including inflammation and metastasis development. CXCR4 also binds the HIV-1 envelope glycoprotein, gp120, resulting in viral entry into host cells. Therefore, CXCR4 and its ligands represent valuable drug targets. In this study, we investigated the inhibitory properties of synthetic peptides derived from CXCR4 extracellular loops (ECL1-X4, ECL2-X4 and ECL3-X4) towards HIV-1 infection and CXCL12-mediated receptor activation. Among these peptides, ECL1-X4 displayed anti-HIV-1 activity against X4, R5/X4 and R5 viruses (IC50=24 to 76µM) in cell viability assay without impairing physiological CXCR4-CXCL12 signalling. In contrast, ECL2-X4 only inhibited X4 and R5/X4 strains, interfering with HIV-entry into cells. At the same time, ECL2-X4 strongly and specifically interacted with CXCL12, blocking its binding to CXCR4 and its second receptor, CXCR7 (IC50=20 and 100µM). Further analysis using mutated and truncated peptides showed that ECL2 of CXCR4 forms multiple contacts with the gp120 protein and the N-terminus of CXCL12. Chemokine neutralisation was mainly driven by four aspartates and the C-terminal residues of ECL2-X4. These results demonstrate that ECL2 represents an important structural determinant in CXCR4 activation. We identified the putative site for the binding of CXCL12 N-terminus and provided new structural elements to explain the recognition of gp120 and dimeric CXCR4 ligands.

Nanoluciferase-based cell fusion assay for rapid and high-throughput assessment of SARS-CoV-2-neutralizing antibodies in patient samples.

After more than two years, COVID-19 still represents a global health burden of unprecedented extent and assessing the degree of immunity of individuals against SARS-CoV-2 remains a challenge. Virus neutralization assays represent the gold standard for assessing antibody-mediated protection against SARS-CoV-2 in sera from recovered and/or vaccinated individuals. Neutralizing antibodies block the interaction of viral spike protein with human angiotensin-converting enzyme 2 (ACE2) receptor in vitro and prevent viral entry into host cells. Classical viral neutralization assays using full replication-competent viruses are restricted to specific biosafety level 3-certified laboratories, limiting their utility for routine and large-scale applications. We developed therefore a cell-fusion-based assay building on the interaction between viral spike and ACE2 receptor expressed on two different cell lines, substantially reducing biosafety risks associated with classical viral neutralization assays. This chapter describes this simple, sensitive, safe and cost-effective approach for rapid and high-throughput evaluation of SARS-CoV-2 neutralizing antibodies relying on high-affinity NanoLuc® luciferase complementation technology (HiBiT). When applied to a variety of standards and patient samples, this method yields highly reproducible results in 96-well, as well as in 384-well format. The use of novel NanoLuc® substrates with increased signal stability like Nano-Glo® Endurazine™ furthermore allows for high flexibility in assay set-up and full automatization of all reading processes. Lastly, the assay is suitable to evaluate the neutralizing capacity of sera against the existing spike variants, and potentially variants that will emerge in the future.

FHR4-based immunoconjugates direct complement-dependent cytotoxicity and phagocytosis towards HER2-positive cancer cells.

Directing selective complement activation towards tumour cells is an attractive strategy to promote their elimination. In the present work, we have generated heteromultimeric immunoconjugates that selectively activate the complement alternative pathway (AP) on tumour cells. We used the C4b-binding protein C-terminal-α-/ß-chain scaffold for multimerisation to generate heteromultimeric immunoconjugates displaying (a) a multivalent-positive regulator of the AP, the human factor H-related protein 4 (FHR4) with; (b) a multivalent targeting function directed against erbB2 (HER2); and (c) a monovalent enhanced GFP tracking function. Two distinct VH H targeting two different epitopes against HER2 and competing either with trastuzumab or with pertuzumab-recognising epitopes [VH H(T) or VH H(P)], respectively, were used as HER2 anchoring moieties. Optimised high-FHR4 valence heteromultimeric immunoconjugates [FHR4/VH H(T) or FHR4/VH H(P)] were selected by sequential cell cloning and a selective multistep His-Trap purification. Optimised FHR4-heteromultimeric immunoconjugates successfully overcame FH-mediated complement inhibition threshold, causing increased C3b deposition on SK-OV-3, BT474 and SK-BR3 tumour cells, and increased formation of lytic membrane attack complex densities and complement-dependent cytotoxicity (CDC). CDC varies according to the pattern expression and densities of membrane-anchored complement regulatory proteins on tumour cell surfaces. In addition, opsonised BT474 tumour cells were efficiently phagocytosed by macrophages through complement-dependent cell-mediated cytotoxicity. We showed that the degree of FHR4-multivalency within the multimeric immunoconjugates was the key element to efficiently compete and deregulate FH and FH-mediated convertase decay locally on tumour cell surface. FHR4 can thus represent a novel therapeutic molecule, when expressed as a multimeric entity and associated with an anchoring system, to locally shift the complement steady-state towards activation on tumour cell surface.

A new recombinant virus system for the study of HIV-1 entry and inhibition.

The construction is described of a HIV-1 proviral, eGFP-tagged plasmid that allows for the recombination of any selected env gene without the use of restriction enzymes and for the quantitation of the infection by the recombinant virus using flow cytometry. The system was tested showing that an isoleucine to valine substitution at residue position 37 of the HIV-1 gp41 impairs the fitness of the virus but does not lead by itself to T-20 resistance.

HIV-1 subtypes in Luxembourg, 1983-2000.

OBJECTIVES: To study the prevalence of HIV-1 subtypes in Luxembourg between 1983 and 2000. To compare the drug susceptibility of non-B and B clade viruses and the prevalence of resistance-associated mutations and polymorphisms before antiretroviral treatment. DESIGN: A retrospective study on plasma samples of HIV-infected patients registered at the National Service of Infectious Diseases, Luxembourg, between 1983 and 2000. METHODS: Genotyping was performed by sequencing of the reverse transcriptase (RT) and protease coding region of the pol gene. Drug susceptibility was assessed in a recombinant virus assay. RESULTS: A total of 20.1% of the HIV-positive patients were infected with non-B subtypes, and since 1990 the proportion of non-B viruses has increased ninefold. Eleven out of 14 F1 subtypes occurred in patients native to Luxembourg. Major resistance mutations related to protease inhibitors (PI), nucleoside reverse transcriptase inhibitors (NRTI) and non-nucleoside reverse transcriptase inhibitors (NNRTI) occurred in less than 3% of treatment-naive viruses; however, 87% of the viruses had at least one PI-associated mutation. Natural polymorphism of the protease and RT coding region was observed more frequently among non-B than B viruses. Significantly more B viruses displayed resistance to the tested PI, NRTI and NNRTI (P = 0.044). CONCLUSION: The proportion of non-B viruses has increased dramatically since 1990. Non-B subtypes showed no decreased susceptibility to antiretroviral drugs, but displayed minor mutations and polymorphisms at higher frequency in their protease and RT coding region. In contrast, a significantly higher proportion of B viruses showed resistance to a range of antiretroviral drugs.

Selection of a CXCR4 antagonist from a human heavy chain CDR3-derived phage library.

Phage display technology is a powerful selection approach to identify strong and specific binders to a large variety of targets. In this study, we compared the efficacy of a phage library displaying human heavy chain complementarity determining region 3 (HCDR3) repertoires with a set of conventional random peptide libraries for the identification of CXCR4 antagonists using a peptide corresponding to the second extracellular loop of the receptor CXCR4 as target. A total of 11 selection campaigns on this target did not result in any specific ligand from the random peptide libraries. In contrast, a single selection campaign with an HCDR3 library derived from the IgM repertoire of a nonimmunized donor resulted in nine specific peptides with lengths ranging from 10 to 19 residues. Four of these HCDR3 sequences interacted with native receptor and the most frequently isolated peptide displayed an affinity of 5.6 µm and acted as a CXCR4 antagonist (IC(50) = 23 µm). To comprehend the basis of the highly efficient HCDR3 library selection, its biochemical properties were investigated. The HCDR3 length varied from 3 to 21 residues and displayed a biased amino acid content with a predominant proportion of Tyr, Gly, Ser and Asp. Repetitive and conserved motifs were observed in the majority of the HCDR3 sequences. The strength and efficacy of the HCDR3 libraries reside in the combination of multiple size peptides and a naturally biased sequence variation. Therefore, HCDR3 libraries represent a powerful and versatile alternative to fully randomized peptide libraries, in particular for difficult targets.

Impact on replicative fitness of the G48E substitution in the protease of HIV-1: an in vitro and in silico evaluation.

We observed an unusual glycine-to-glutamate substitution at protease (PR) residue position 48 (G48E) in an African patient infected with a subtype A1 HIV-1 strain failing a saquinavir-containing regimen. Phenotypic analysis of protease inhibitor (PI) susceptibility showed that the G48E site-directed mutant, when introduced into an NL4-3 HIV-1 PR backbone, was slightly resistant to SQV (2-fold when compared with the wild-type virus). In addition, the G48E and G48E/V82A site-directed mutants were associated with a decrease in fitness, whereas a reversion to the wild type at position 48 was observed in vitro. Growth competition experiments using a novel growth competition assay based on enhanced green fluorescent protein- or Discosoma spp. red fluorescent protein-expressing viruses showed that the replicative fitness of the G48E virus was reduced to 55% compared with the parental NL4-3 virus. Synthesizing all possible site-directed mutants found in the patient strain is too time-consuming; therefore, a molecular dynamics (MD) simulation approach was used to understand why this mutation survived despite its fitness cost. These simulations documented that the G48E mutant interacted with PI resistance mutations (M46I, I54V, Q58E, and L63P) and with natural polymorphisms specific to subtype A1 (E35D, M36I, and R57K) that were present in the patient's virus. We hypothesize that the polymorphisms contained in the PR flap regions of the patient's virus may compensate for the presence of G48E, possibly by restoring the flexibility of the PR flaps. In summary, our results demonstrate that the G48E substitution, when introduced in the context of an HIV-1 subtype B strain, is highly unstable and gives rise to viruses with a poor replicative fitness in vitro. We also showed that when confronted with too many mutations to evaluate in vitro, MD simulations are helpful to draft hypotheses on how polymorphisms can interact with resistance mutations to stabilize their potential fitness cost.

Longitudinal use of phenotypic resistance testing to HIV-1 protease inhibitors in patients developing HAART failure.

An "in-house" recombinant virus protease inhibitor susceptibility assay was carried out (median of 3 per patient) retrospectively in 26 patients failing HIV protease inhibitor based therapy at regular intervals to the initiation of the first protease inhibitor. Patients were treated with either indinavir (N = 6), ritonavir (N = 10), or saquinavir (N = 10) and two nucleoside analogues. Second line therapy was based on single or dual protease inhibitor regimens occasionally containing nelfinavir. Clinically relevant resistance cut-offs associated with a poorer virological outcome from 6 months on and the clinical outcome from 3 months on were determined tentatively as 4- to 8-fold resistance for indinavir and ritonavir and 2.5- to 8-fold to saquinavir. In addition, the degree of cross-resistance at the time of the change of protease inhibitor was associated with the response in viral load at 6 months to the second line therapy (P = 0.018). Cross-resistance (> or = 8-fold) between ritonavir and indinavir was common (78 and 100%). Cross-resistance between indinavir or ritonavir and saquinavir was less frequent (75 and 60% respectively) than the opposite (100%, P = 0.004). Cross-resistance to nelfinavir was encountered more frequently (> 70%) than to amprenavir (9%). The magnitudes of resistance were correlated between each other. In summary, the protease inhibitor susceptibility carried out longitudinally appears to be an earlier prognostic marker than viral load in a context of cross-resistance. The magnitude of resistance, as a marker of cross-resistance, should be useful to guide second line therapy.

Longitudinal use of a line probe assay for human immunodeficiency virus type 1 protease predicts phenotypic resistance and clinical progression in patients failing highly active antiretroviral therapy.

An observational study assessed the longitudinal use of a new line probe assay for the detection of protease mutations. Probe assays for detection of reverse transcriptase (Inno-LiPA HIV-1 RT; Innogenetics) and protease (prototype kit Inno-LiPA HIV Protease; Innogenetics) mutations gave results for 177 of 199 sequential samples collected over 2 years from 26 patients failing two nucleoside reverse transcriptase inhibitors and one protease inhibitor (first line: indinavir, n = 6; ritonavir, n = 10; and saquinavir, n = 10). Results were compared to recombinant virus protease inhibitor susceptibility data (n = 87) and to clinical and virological data. Combinations of protease mutations (M46I, G48V, I54V, V82A or -F, I84V, and L90M) predicted phenotypic resistance to the protease inhibitor and to nelfinavir. The sum of protease mutations was associated with virological and clinical outcomes from 6 and 3 months on, respectively. Moreover, a poorer clinical outcome was linked to the sum of reverse transcriptase mutations. In conclusion, despite the limited number of patients studied and the restricted number of codons investigated, probe assay-based genotyping correlates with phenotypic drug resistance and predicts new Centers for Disease Control and Prevention stage B and C clinical events and virological outcome. Line probe assays provide additional prognostic information and should be prospectively investigated for their potential for treatment monitoring.