Systematic Evaluation of Fluorination as Modification for Peptide‐Based Fusion Inhibitors against HIV‐1 Infection

With the emergence of novel viruses, the development of new antivirals is more urgent than ever. A key step in human immunodeficiency virus type 1 (HIV-1) infection is six-helix bundle formation within the envelope protein subunit gp41. Selective disruption of bundle formation by peptides has been shown to be effective; however, these drugs, exemplified by T20, are prone to rapid clearance from the patient. The incorporation of non-natural amino acids is known to improve these pharmacokinetic properties. Here, we evaluate a peptide inhibitor in which a critical Ile residue is replaced by fluorinated analogues. We characterized the influence of the fluorinated analogues on the biophysical properties of the peptide. Furthermore, we show that the fluorinated peptides can block HIV-1 infection of target cells at nanomolar levels. These findings demonstrate that fluorinated amino acids are appropriate tools for the development of novel peptide therapeutics

Targeted escape of SARS-CoV-2 in vitro from monoclonal antibody S309, the precursor of sotrovimab

Class 1 and 2 monoclonal antibodies inhibit SARS-CoV-2 entry by blocking the interaction of the viral receptor-binding domain with angiotensin-converting enzyme 2 (ACE2), while class 3 antibodies target a highly conserved epitope outside the ACE2 binding site. We aimed to investigate the plasticity of the spike protein by propagating wild-type SARS-CoV-2 in the presence of class 3 antibody S309. After 12 weeks, we obtained a viral strain that was completely resistant to inhibition by S309, due to successively evolving amino acid exchanges R346S and P337L located in the paratope of S309. The antibody lost affinity to receptor-binding domains carrying P337L or both amino acid exchanges, while ACE2 binding was not affected. The resistant strain replicated efficiently in human CaCo-2 cells and was more susceptible to inhibition of fusion than the original strain. Overall, SARS-CoV-2 escaped inhibition by class 3 antibody S309 through a slow, but targeted evolution enabling immune escape and altering cell entry. This immune-driven enhancement of infectivity and pathogenicity could play an important role in the future evolution of SARSCoV-2, which is under increasing immunological pressure from vaccination and previous infections

Generation of an Oncolytic Herpes Simplex Virus 1 Expressing Human MelanA

Robust anti-tumor immunity requires innate as well as adaptive immune responses. We have shown that plasmacytoid dendritic cells develop killer cell-like activity in melanoma cell cocultures after exposure to the infectious but replication-deficient herpes simplex virus 1 (HSV-1) d106S. To combine this innate effect with an enhanced adaptive immune response, the gene encoding human MelanA/MART-1 was inserted into HSV-1 d106S via homologous recombination to increase direct expression of this tumor antigen. Infection of Vero cells using this recombinant virus confirmed MelanA expression by Western blotting, flow cytometry, and immunofluorescence. HSV-1 d106S-MelanA induced expression of the transgene in fibroblast and melanoma cell lines not naturally expressing MelanA. Infection of a melanoma cell line with CRISPR-Cas9-mediated knockout of MelanA confirmed de novo expression of the transgene in the viral context. Dependent on MelanA expression, infected fibroblast and melanoma cell lines induced degranulation of HLA-matched MelanA-specific CD8+ T cells, followed by killing of infected cells. To study infection of immune cells, we exposed peripheral blood mononuclear cells and in vitro-differentiated macrophages to the parental HSV-1 d106S, resulting in expression of the transgene GFP in CD11c+ cells and macrophages. These data provide evidence that the application of MelanA-encoding HSV-1 d106S could enhance adaptive immune responses and re-direct MelanA-specific CD8+ T cells to tumor lesions, which have escaped adaptive immune responses via downregulation of their tumor antigen. Hence, HSV-1 d106S-MelanA harbors the potential to induce innate immune responses in conjunction with adaptive anti-tumor responses by CD8+ T cells, which should be evaluated in further studies

Nectin-1 Expression Correlates with the Susceptibility of Malignant Melanoma to Oncolytic Herpes Simplex Virus In Vitro and In Vivo

Talimogene laherparepvec (T-VEC), an oncolytic herpes simplex virus, is approved for intralesional injection of unresectable stage IIIB/IVM1a melanoma. However, it is still unclear which parameter(s) predict treatment response or failure. Our study aimed at characterizing surface receptors Nectin-1 and the herpes virus entry mediator (HVEM) in addition to intracellular molecules cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) as potential bio-markers for oncolytic virus treatment. In 20 melanoma cell lines, oncolytic activity of T-VEC was correlated with the expression of Nectin-1 but not HVEM, as evaluated via flow cytometry and immunohistochemistry. Knockout using CRISPR/Cas9 technology confirmed the superior role of Nectin-1 over HVEM for entry and oncolytic activity of T-VEC. Neither cGAS nor STING as evaluated by Western Blot and immunohistochemistry correlated with T-VEC induced oncolysis. The role of these biomarkers was retrospectively analyzed for the response of 35 cutaneous melanoma metastases of 21 patients to intralesional T-VEC injection, with 21 (60.0%) of these lesions responding with complete (n = 16) or partial regression (n = 5). Nectin-1 expression in pretreatment biopsies significantly predicted treatment outcome, while the expression of HVEM, cGAS, and STING was not prognostic. Altogether, Nectin-1 served as biomarker for T-VEC-induced melanoma regression in vitro and in vivo

Comparison of the oncolytic activity of a replication‐competent and a replication‐deficient herpes simplex virus 1

In 2015, the oncolytic herpes simplex virus 1 (HSV-1) T-VEC (talimogene laherparepvec) was approved for intratumoral injection in non-resectable malignant melanoma. To determine whether viral replication is required for oncolytic activity, we compared replication-deficient HSV-1 d106S with replication-competent T-VEC. High infectious doses of HSV-1 d106S killed melanoma (n = 10), head-and-neck squamous cell carcinoma (n = 11), and chondrosarcoma cell lines (n = 2) significantly faster than T-VEC as measured by MTT metabolic activity, while low doses of T-VEC were more effective over time. HSV-1 d106S and, to a lesser extent T-VEC, triggered caspase-dependent early apoptosis as shown by pan-caspase inhibition and specific induction of caspases 3/7, 8, and 9. HSV-1 d106S induced a higher ratio of apoptosis-inducing infected cell protein (ICP) 0 to apoptosis-blocking ICP6 than T-VEC. T-VEC was oncolytic for an extended period of time as viral replication continued, which could be partially blocked by the antiviral drug aciclovir. High doses of T-VEC, but not HSV-1 d106S, increased interferon-β mRNA as part of the intrinsic immune response. When markers of immunogenic cell death were assessed, ATP was released more efficiently in the context of T-VEC than HSV-1 d106S infection, whereas HMGB1 was induced comparatively well. Overall, the early oncolytic effect on three different tumour entities was stronger with the non-replicative strain, while the replication-competent virus elicited a stronger innate immune response and more pronounced immunogenic cell death

Exploring Viral Interference Using Peptides: Molecular Determinants of HIV‐1 Inhibition by a Peptide Derived from Human Pegivirus‐1 Envelope Protein E2

Co-infection with the human pegivirus 1 (HPgV-1) often has a beneficial effect on disease progression in HIV-1-infected individuals. Several HPgV-1 proteins and peptides, including a 20-mer peptide (P6-2) derived from the N-terminal region of the HPgV-1 surface protein E2, have been associated with this phenomenon, which is referred to as viral interference. We identified the cysteine residues, the hydrophobic core tetrapeptide, as well as the C-terminal negative charge as key factors for the HIV-1 inhibitory activity of P6-2. Analysis of mutations in P6-2-resistant HIV-1 indicated a binding site for the peptide in the HIV-1 envelope glycoprotein gp120. In fact, P6-2 was shown to bind to soluble gp120, as well as to a peptide presenting the gp120 V3 loop. Furthermore, the HIV-1 inhibitory activity of P6-2 could be revoked by the V3 loop peptide, thus indicating a molecular mechanism that involves interaction of P6-2 with the gp120 V3 loop

Systematic Evaluation of Fluorination as Modification for Peptide-Based Fusion Inhibitors against HIV-1 Infection

With the emergence of novel viruses, the development of new antivirals is more urgent than ever. A key step in human immunodeficiency virus type 1 (HIV-1) infection is six-helix bundle formation within the envelope protein subunit gp41. Selective disruption of bundle formation by peptides has been shown to be effective; however, these drugs, exemplified by T20, are prone to rapid clearance from the patient. The incorporation of non-natural amino acids is known to improve these pharmacokinetic properties. Here, we evaluate a peptide inhibitor in which a critical Ile residue is replaced by fluorinated analogues. We characterized the influence of the fluorinated analogues on the biophysical properties of the peptide. Furthermore, we show that the fluorinated peptides can block HIV-1 infection of target cells at nanomolar levels. These findings demonstrate that fluorinated amino acids are appropriate tools for the development of novel peptide therapeutics

Inhibition of HIV-1 infection by human pegivirus type 1-derived peptides is affected by human pegivirus type 1 genotype and HIV-1 coreceptor tropism

Objective(s): Up to 40% of HIV-1 infected individuals are coinfected with human pegivirus type 1 (HPgV-1). The majority of studies, but not all, have reported a beneficial effect of HPgV-1 coinfection on HIV-1 disease progression. So far, the impact of different HPgV-1 genotypes on different HIV-1 subtypes remains unclear. Methods: Peptides derived from HPgV-1 envelope protein E2, and representing different viral genotypes, were synthesized using Fmoc/t-Bu-based solid phase peptide synthesis. The inhibitory effect of these peptides on the infection of reporter cell lines was tested using an HIV-1 subtype panel representing clades A (n = 2), AG (n = 2), B (n = 6), C (n = 2), D (n = 2), F (n = 2), G (n = 1), G/H (n = 1), and group O (n = 2). Results: HIV-1 infection was blocked more efficiently by peptides derived from HPgV-1 GT2 than GT1 (P = 0.05). The HIV-1 subtype did not affect the degree of inhibition by a peptide derived from HPgV-1 GT2. All CXCR4-/dual-tropic isolates (n = 12), but only half (four out of eight) CCR5-tropic viruses were inhibited by this peptide (P = 0.014). Conclusion: Our data indicate that the inhibitory effect of peptides derived from HPgV-1 E2 protein is dependent on the genotype, suggesting that coinfection with HPgV-1 GT1 is less likely to confer a beneficial effect on HIV-1 disease progression than GT2. The preferential suppression of more pathogenic CXCR4-tropic HIV-1 by peptides derived from HPgV-1 GT2 may explain the favorable effect in patients harboring these HIV-1 isolates. Consequently, HPgV-1 genotype and HIV-1 coreceptor tropism are likely determinants for the beneficial effect of HPgV-1 co-infection in HIV-1-infected individuals. Copyright (C) 2018 Wolters Kluwer Health, Inc. All rights reserved