Lineage Replacement and Genetic Changes of Four HR-HPV Types during the Period of Vaccine Coverage: A Six-Year Retrospective Study in Eastern China.

OBJECTIVE: This study aimed to provide clinical evidence for lineage replacement and genetic changes of High-Risk Human Papillomavirus (HR-HPV) during the period of vaccine coverage and characterize those changes in eastern China. METHODS: This study consisted of two stages. A total of 90,583 patients visiting the Obstetrics and Gynecology Hospital of Fudan University from March 2018 to March 2022 were included in the HPV typing analysis. Another 1076 patients who tested positive for HPV31, 33, 52, or 58 from November 2020 to August 2023 were further included for HPV sequencing. Vaccination records, especially vaccine types and the third dose administration time, medical history, and cervical cytology samples were collected. Viral DNA sequencing was then conducted, followed by phylogenetic analysis and sequence alignment. RESULTS: The overall proportion of HPV31 and 58 infections increased by 1.23% and 0.51%, respectively, while infection by HPV33 and 52 decreased by 0.42% and 1.43%, respectively, within the four-year vaccination coverage period. The proportion of HPV31 C lineage infections showed a 22.17% increase in the vaccinated group, while that of the HPV58 A2 sublineage showed a 12.96% increase. T267A and T274N in the F-G loop of HPV31 L1 protein, L150F in the D-E loop, and T375N in the H-I loop of HPV58 L1 protein were identified as high-frequency escape-related mutations. CONCLUSIONS: Differences in epidemic lineage changes and dominant mutation accumulation may result in a proportional difference in trends of HPV infection. New epidemic lineages and high-frequency escape-related mutations should be noted during the vaccine coverage period, and regional epidemic variants should be considered during the development of next-generation vaccines.

Structure-based design of pan-coronavirus inhibitors targeting host cathepsin L and calpain-1.

Respiratory disease caused by coronavirus infection remains a global health crisis. Although several SARS-CoV-2-specific vaccines and direct-acting antivirals are available, their efficacy on emerging coronaviruses in the future, including SARS-CoV-2 variants, might be compromised. Host-targeting antivirals provide preventive and therapeutic strategies to overcome resistance and manage future outbreak of emerging coronaviruses. Cathepsin L (CTSL) and calpain-1 (CAPN1) are host cysteine proteases which play crucial roles in coronaviral entrance into cells and infection-related immune response. Here, two peptidomimetic α-ketoamide compounds, 14a and 14b, were identified as potent dual target inhibitors against CTSL and CAPN1. The X-ray crystal structures of human CTSL and CAPN1 in complex with 14a and 14b revealed the covalent binding of α-ketoamide groups of 14a and 14b to C25 of CTSL and C115 of CAPN1. Both showed potent and broad-spectrum anticoronaviral activities in vitro, and it is worth noting that they exhibited low nanomolar potency against SARS-CoV-2 and its variants of concern (VOCs) with EC50 values ranging from 0.80 to 161.7 nM in various cells. Preliminary mechanistic exploration indicated that they exhibited anticoronaviral activity through blocking viral entrance. Moreover, 14a and 14b exhibited good oral pharmacokinetic properties in mice, rats and dogs, and favorable safety in mice. In addition, both 14a and 14b treatments demonstrated potent antiviral potency against SARS-CoV-2 XBB 1.16 variant infection in a K18-hACE2 transgenic mouse model. And 14b also showed effective antiviral activity against HCoV-OC43 infection in a mouse model with a final survival rate of 60%. Further evaluation showed that 14a and 14b exhibited excellent anti-inflammatory effects in Raw 264.7 mouse macrophages and in mice with acute pneumonia. Taken together, these results suggested that 14a and 14b are promising drug candidates, providing novel insight into developing pan-coronavirus inhibitors with antiviral and anti-inflammatory properties.

gp120-derived amyloidogenic peptides form amyloid fibrils that increase HIV-1 infectivity.

Apart from mediating viral entry, the function of the free HIV-1 envelope protein (gp120) has yet to be elucidated. Our group previously showed that EP2 derived from one ß-strand in gp120 can form amyloid fibrils that increase HIV-1 infectivity. Importantly, gp120 contains ~30 ß-strands. We examined whether gp120 might serve as a precursor protein for the proteolytic release of amyloidogenic fragments that form amyloid fibrils, thereby promoting viral infection. Peptide array scanning, enzyme degradation assays, and viral infection experiments in vitro confirmed that many ß-stranded peptides derived from gp120 can indeed form amyloid fibrils that increase HIV-1 infectivity. These gp120-derived amyloidogenic peptides, or GAPs, which were confirmed to form amyloid fibrils, were termed gp120-derived enhancers of viral infection (GEVIs). GEVIs specifically capture HIV-1 virions and promote their attachment to target cells, thereby increasing HIV-1 infectivity. Different GAPs can cross-interact to form heterogeneous fibrils that retain the ability to increase HIV-1 infectivity. GEVIs even suppressed the antiviral activity of a panel of antiretroviral agents. Notably, endogenous GAPs and GEVIs were found in the lymphatic fluid, lymph nodes, and cerebrospinal fluid (CSF) of AIDS patients in vivo. Overall, gp120-derived amyloid fibrils might play a crucial role in the process of HIV-1 infectivity and thus represent novel targets for anti-HIV therapeutics.

Design of MERS-CoV entry inhibitory short peptides based on helix-stabilizing strategies.

Interaction between Middle East respiratory syndrome coronavirus (MERS-CoV) spike (S) protein heptad repeat-1 domain (HR1) and heptad repeat-2 domain (HR2) is critical for the MERS-CoV fusion process. This interaction is mediated by the α-helical region from HR2 and the hydrophobic groove in a central HR1 trimeric coiled coil. We sought to develop a short peptidomimetic to act as a MERS-CoV fusion inhibitor by reproducing the key recognition features of HR2 helix. This was achieved by the use of helix-stabilizing strategies, including substitution with unnatural helix-favoring amino acids, introduction of ion pair interactions, and conjugation of palmitic acid. The resulting 23-mer lipopeptide, termed AEEA-C16, inhibits MERS-CoV S protein-mediated cell-cell fusion at a low micromolar level comparable to that of the 36-mer HR2 peptide HR2P-M2. Collectively, our studies provide new insights into developing short peptide-based antiviral agents to treat MERS-CoV infection.

Repurposing Navitoclax to block SARS-CoV-2 fusion and entry by targeting heptapeptide repeat sequence 1 in S2 protein.

Along with the long pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has come the dilemma of emerging viral variants of concern (VOC), particularly Omicron and its subvariants, able to deftly escape immune surveillance and the otherwise protective effect of current vaccines and antibody drugs. We previously identified a peptide-based pan-CoV fusion inhibitor, termed as EK1, able to bind the HR1 region in viral spike (S) protein S2 subunit. This effectively blocked formation of the six-helix bundle (6-HB) fusion core and, thus, showed efficacy against all human coronaviruses (HCoVs). EK1 is now in phase 3 clinical trials. However, the peptide drug generally lacks oral availability. Therefore, we herein performed a structure-based virtual screening of the libraries of biologically active molecules and identified nine candidate compounds. One is Navitoclax, an orally active anticancer drug by inhibition of Bcl-2. Like EK1 peptide, it could bind HR1 and block 6-HB formation, efficiently inhibiting fusion and infection of all SARS-CoV-2 variants tested, as well as SARS-CoV and MERS-CoV, with IC50 values ranging from 0.5 to 3.7 µM. These findings suggest that Navitoclax is a promising repurposed drug candidate for development as a safe and orally available broad-spectrum antiviral drug to combat the current SARS-CoV-2 and its variants, as well as other HCoVs.

25-hydroxycholesterol inhibits human papillomavirus infection in cervical epithelial cells by perturbing cytoskeletal remodeling.

Persistent high-risk human papilloma virus (HR-HPV) infection is the main risk factor for cervical cancer, threatening women's health. Despite growing prophylactic vaccination, annual cervical cancer cases are still increasing and show a trend of younger onset age. However, therapeutic approaches towards HPV infection are still limited. 25-hydrocholesterol (25HC) has a wide-spectrum inhibitory effect on a variety of viruses. To explore efficient interventions to restrict HPV infection at an early time, we applied different pseudoviruses (PsV) to evaluate anti-HPV efficacy of 25HC. We tested PsV inhibition by 25HC in cervical epithelial-derived HeLa and C-33A cells, using high-risk (HPV16, HPV18, HPV59), possibly carcinogenic (HPV73), and low-risk (HPV6) HPV PsVs. Then we established murine genital HPV PsV infection models and applied IVIS to evaluate anti-HPV efficacy of 25HC in vivo. Next, with the help of confocal imaging, we targeted 25HC activity at filopodia upon HPV exposure. After that, we used RNA-seq and Western blot analysis to investigate (1) how 25HC disturbs actin cytoskeleton remodeling during HPV infection and (2) how prenylation regulates the cytoskeletal remodeling signaling pathway. Our findings suggest that 25HC perturbs F-actin rearrangement by reducing small GTPase prenylation. In this way, the phenomenon of HPV virion surfing was restricted, leading to failed infection.

An Artificial Peptide-Based Bifunctional HIV-1 Entry Inhibitor That Interferes with Viral Glycoprotein-41 Six-Helix Bundle Formation and Antagonizes CCR5 on the Host Cell Membrane.

Human immunodeficiency virus type 1 (HIV-1) is characterized by high variability and drug resistance. This has necessitated the development of antivirals with a new chemotype and therapy. We previously identified an artificial peptide with non-native protein sequence, AP3, with the potential to inhibit HIV-1 fusion through targeting hydrophobic grooves on the N-terminal heptad repeat trimer of viral glycoprotein gp41. Here, a small-molecule HIV-1 inhibitor targeting chemokine coreceptor CCR5 on the host cell was integrated into the AP3 peptide, producing a novel dual-target inhibitor with improved activity against multiple HIV-1 strains including those resistant to the currently used anti-HIV-1 drug enfuvirtide. Its superior antiviral potency in comparison with the respective pharmacophoric moieties is in consonance with the dual binding of viral gp41 and host factor CCR5. Therefore, our work provides a potent artificial peptide-based bifunctional HIV-1 entry inhibitor and highlights the multitarget-directed ligands approach in the development of novel therapeutic anti-HIV-1 agents.

Multitargeted drug design strategy for discovery of short-peptide-based HIV-1 entry inhibitors with high potency.

The development of short-peptide-based inhibitors to prevent HIV-1 entry into the host cell has been rewarded with limited success. Herein, we report a multitarget-directed ligand strategy to generate a series of short-peptide HIV-1 entry inhibitors that integrated the pharmacological activities of a peptide fusion inhibitor able to disrupt HIV-1 gp41 glycoprotein hexameric coiled-coil assembly and a small-molecule CCR5 antagonist that blocks the interaction between HIV-1 and its coreceptor. Among these inhibitors, dual-target 23-residue peptides SP12T and SP12L displayed dramatically increased inhibitory activities against HIV-1 replication as compared to the marketed 36-residue peptide T20. Moreover, results suggested that SP12T and SP12L successfully performed a dual-targeting mechanism. It can be concluded that these short-peptide-based HIV-1 entry inhibitors have potential for further development as candidates for a novel multitarget therapy to treat HIV-1 infection.

Chemically Modified Bovine ß-Lactoglobulin as a Broad-Spectrum Influenza Virus Entry Inhibitor with the Potential to Combat Influenza Outbreaks.

Frequent outbreaks of the highly pathogenic influenza A virus (AIV) infection, together with the lack of broad-spectrum influenza vaccines, call for the development of broad-spectrum prophylactic agents. Previously, 3-hydroxyphthalic anhydride-modified bovine ß-lactoglobulin (3HP-ß-LG) was proven to be effective against human immunodeficiency virus (HIV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and it has also been used in the clinical control of cervical human papillomavirus (HPV) infections. Here, we show its efficacy in potently inhibiting infection by divergent influenza A and B viruses. Mechanistic studies suggest that 3HP-ß-LG binds, possibly through its negatively charged residues, to the receptor-binding domain in the hemagglutinin 1 (HA1) subunit in the HA of the influenza virus, thus inhibiting the attachment of the HA to sialic acid on host cells. The intranasal administration of 3HP-ß-LG led to the protection of mice against challenges by influenza A(H1N1)/PR8, A(H3N2), and A(H7N9) viruses. Furthermore, 3HP-ß-LG is highly stable when stored at 50 °C for 30 days and it shows excellent safety in vitro and in vivo. Collectively, our findings suggest that 3HP-ß-LG could be successfully repurposed as an intranasal prophylactic agent to prevent influenza virus infections during influenza outbreaks.

Design of artificial α-helical peptides targeting both gp41 deep pocket and subpocket as potent HIV-1 fusion inhibitors.

Both the deep pocket region and its neighboring subpocket site on the N-trimer of HIV-1 gp41 protein can serve as targets for the development of HIV-1 entry inhibitors. Pocket-binding domain (PBD)-containing peptides with the potential to inhibit HIV-1 fusion through targeting the deep pocket have been extensively exploited. However, using an artificial peptide strategy, we herein report the design of α-helical lipopeptides with non-native protein sequences as HIV-1 fusion inhibitors that can occupy both gp41 deep cavity and subpocket sites. The most active compound, PP24C, inhibited HIV-1 replication, including T20-resistant HIV-1 mutants, at low nanomolar level. Biophysical approaches revealed that both the artificial α-helical peptide P35A4 and its cholesterol-tagged peptide PP24C could bind to T21 peptide used as a target surrogate comprising both pockets. Our study offers a new template for the design of artificial anti-HIV-1 therapeutics and highlights the novel concept of peptide secondary structure-based virus fusion inhibitors.

Virus Entry Inhibitors: Past, Present, and Future.

The approval of enfuvirtide marked a milestone for the development of virus entry inhibitor-based antiviral therapeutics. Since then, more peptide-, small-molecule-, and protein-based entry inhibitors have been identified and approved for viral diseases. Here we reviewed the development of virus entry inhibitors and the advantages and disadvantages of peptide-, small-molecule-, and protein-based entry inhibitors, herein summarizing the future trend of these antivirals. Virus entry inhibitors take effect outside the host cell, making them good candidates for development as pre- and post-exposure prophylaxis, microbicides, and therapeutics. This chapter, as well as this book, provides more information on the development and modification of peptide-, small-molecule-, and protein-based virus entry inhibitors.

Peptide-Based HIV Entry Inhibitors.

The development of peptide-based HIV entry inhibitors has made an important contribution to the stock of anti-HIV drugs. In particular, the peptide-based anti-HIV drugs enfuvirtide and albuvirtide were approved for clinical use by the U.S. FDA and CFDA in 2003 and 2018, respectively. Peptide-based HIV entry inhibitors exert antiviral activity by targeting the early stage of viral infection, i.e., binding of a viral surface protein to the receptor(s) on the host cell and the subsequent fusion between the viral and host cell membranes. Therefore, they are particularly useful for HIV-infected patients who have failed to respond to the highly active antiretroviral drugs (ARD) targeting the late stage of HIV replication, such as reverse transcriptase inhibitors and protease inhibitors. In this chapter, we will focus on the past, current, and future trends in research and development of peptide-based HIV entry inhibitors.

A Five-Helix-Based SARS-CoV-2 Fusion Inhibitor Targeting Heptad Repeat 2 Domain against SARS-CoV-2 and Its Variants of Concern.

The prolonged duration of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic has resulted in the continuous emergence of variants of concern (VOC, e.g., Omicron) and variants of interest (VOI, e.g., Lambda). These variants have challenged the protective efficacy of current COVID-19 vaccines, thus calling for the development of novel therapeutics against SARS-CoV-2 and its VOCs. Here, we constructed a novel fusion inhibitor-based recombinant protein, denoted as 5-Helix, consisting of three heptad repeat 1 (HR1) and two heptad repeat 2 (HR2) fragments. The 5-Helix interacted with the HR2 domain of the viral S2 subunit, the most conserved region in spike (S) protein, to block homologous six-helix bundle (6-HB) formation between viral HR1 and HR2 domains and, hence, viral S-mediated cell-cell fusion. The 5-Helix potently inhibited infection by pseudotyped SARS-CoV-2 and its VOCs, including Delta and Omicron variants. The 5-Helix also inhibited infection by authentic SARS-CoV-2 wild-type (nCoV-SH01) strain and its Delta variant. Collectively, our findings suggest that 5-Helix can be further developed as either a therapeutic or prophylactic to treat and prevent infection by SARS-CoV-2 and its variants.

Viral Entry Inhibitors Targeting Six-Helical Bundle Core against Highly Pathogenic Enveloped Viruses with Class I Fusion Proteins.

Type Ⅰ enveloped viruses bind to cell receptors through surface glycoproteins to initiate infection or undergo receptor-mediated endocytosis and initiate membrane fusion in the acidic environment of endocytic compartments, releasing genetic material into the cell. In the process of membrane fusion, envelope protein exposes fusion peptide, followed by an insertion into the cell membrane or endosomal membrane. Further conformational changes ensue in which the type 1 envelope protein forms a typical six-helix bundle structure, shortening the distance between viral and cell membranes so that fusion can occur. Entry inhibitors targeting viral envelope proteins, or host factors, are effective antiviral agents and have been widely studied. Some have been used clinically, such as T20 and Maraviroc for human immunodeficiency virus 1 (HIV-1) or Myrcludex B for hepatitis D virus (HDV). This review focuses on entry inhibitors that target the six-helical bundle core against highly pathogenic enveloped viruses with class I fusion proteins, including retroviruses, coronaviruses, influenza A viruses, paramyxoviruses, and filoviruses.

Supercoiling Structure-Based Design of a Trimeric Coiled-Coil Peptide with High Potency against HIV-1 and Human ß-Coronavirus Infection.

Hexameric structure formation through packing of three C-terminal helices and an N-terminal trimeric coiled-coil core has been proposed as a general mechanism of class I enveloped virus entry. In this process, the C-terminal helical repeat (HR2) region of viral membrane fusion proteins becomes transiently exposed and accessible to N-terminal helical repeat (HR1) trimer-based fusion inhibitors. Herein, we describe a mimetic of the HIV-1 gp41 HR1 trimer, N3G, as a promising therapeutic against HIV-1 infection. Surprisingly, we found that in addition to protection against HIV-1 infection, N3G was also highly effective in inhibiting infection of human ß-coronaviruses, including MERS-CoV, HCoV-OC43, and SARS-CoV-2, possibly by binding the HR2 region in the spike protein of ß-coronaviruses to block their hexameric structure formation. These studies demonstrate the potential utility of anti-HIV-1 HR1 peptides in inhibiting human ß-coronavirus infection. Moreover, this strategy could be extended to the design of broad-spectrum antivirals based on the supercoiling structure of peptides.

Inhibition of viral suppressor of RNAi proteins by designer peptides protects from enteroviral infection in vivo.

RNA interference (RNAi) is the major antiviral mechanism in plants and invertebrates, but the absence of detectable viral (v)siRNAs in mammalian cells upon viral infection has questioned the functional relevance of this pathway in mammalian immunity. We designed a series of peptides specifically targeting enterovirus A71 (EV-A71)-encoded protein 3A, a viral suppressor of RNAi (VSR). These peptides abrogated the VSR function of EV-A71 in infected cells and resulted in the accumulation of vsiRNAs and reduced viral replication. These vsiRNAs were functional, as evidenced by RISC-loading and silencing of target RNAs. The effects of VSR-targeting peptides (VTPs) on infection with EV-A71 as well as another enterovirus, Coxsackievirus-A16, were ablated upon deletion of Dicer1 or AGO2, core components of the RNAi pathway. In vivo, VTP treatment protected mice against lethal EV-A71 challenge, with detectable vsiRNAs. Our findings provide evidence for the functional relevance of RNAi in mammalian immunity and present a therapeutic strategy for infectious disease.

A safe and effective mucosal RSV vaccine in mice consisting of RSV phosphoprotein and flagellin variant.

Respiratory syncytial virus (RSV) is a major cause of serious acute lower respiratory tract infection in infants and the elderly. The lack of a licensed RSV vaccine calls for the development of vaccines with other targets and vaccination strategies. Here, we construct a recombinant protein, designated P-KFD1, comprising RSV phosphoprotein (P) and the E.-coli-K12-strain-derived flagellin variant KFD1. Intranasal immunization with P-KFD1 inhibits RSV replication in the upper and lower respiratory tract and protects mice against lung disease without vaccine-enhanced disease (VED). The P-specific CD4+ T cells provoked by P-KFD1 intranasal (i.n.) immunization either reside in or migrate to the respiratory tract and mediate protection against RSV infection. Single-cell RNA sequencing (scRNA-seq) and carboxyfluorescein succinimidyl ester (CFSE)-labeled cell transfer further characterize the Th1 and Th17 responses induced by P-KFD1. Finally, we find that anti-viral protection depends on either interferon-γ (IFN-γ) or interleukin-17A (IL-17A). Collectively, P-KFD1 is a promising safe and effective mucosal vaccine candidate for the prevention of RSV infection.