A clinically used anti-human papilloma virus agent (3-hydroxyphthalic anhydride-modified bovine ß-lactoglobulin) has a potential for topical application to prevent sexual transmission of monkeypox virus.

A global outbreak of monkeypox (mpox) caused by the mpox virus (MPXV) has posed a serious threat to public health worldwide, thus calling for the urgent development of antivirals and vaccines to curb its further spread. In this study, we screened 41 anhydride-modified proteins and found that 3-hydroxyphthalic anhydride-modified ß-lactoglobulin (3HP-ß-LG), a clinically used anti-HPV agent, was highly effective in inhibiting infection of vaccinia virus Tiantan strain (VACV-VTT) and MPXV. Mechanistic studies demonstrated that 3HP-ß-LG bound to the virus, not the host cell, by targeting the early stage of virus entry, possibly through the interaction between the amino acids with negatively charges in 3HP-ß-LG and the key amino acids with positive charges in the target region of A29L, a key surface protein of MPXV. A synergistic effect was observed when 3HP-ß-LG was combined with tecovirimat, a small-molecule antiviral drug approved by the United States Food and Drug Administration and the European Medicine Agency for the treatment of smallpox and mpox. Because of its clinically proven safety and stability, 3HP-ß-LG shows promise for further development as a prophylactic agent to prevent the sexual transmission of MPXV.

ADS-J21 is a novel HIV-1 entry inhibitor targeting gp41.

HIV-1 envelope glycoprotein gp41 mediates fusion between HIV-1 and host cell membranes, making inhibitors of gp41 attractive anti-HIV drugs. We previously reported an efficient HIV-1 fusion inhibitor, ADS-J1, with a Y-shaped structure. Here, we discovered a new compound, ADS-J21, with a Y-shaped structure similar to that of ADS-J1 but with a lower molecular weight. Moreover, ADS-J21 exhibited effective anti-HIV-1 activity against divergent HIV-1 strains in vitro, including several HIV-1 laboratory-adapted strains and primary isolates with different subtypes (clades A to F) and tropisms (X4 or R5). Mechanistic studies have demonstrated that ADS-J21 blocks the formation of the gp41 six-helix bundle (6-HB) by targeting conserved amino acids Lys35 and Trp32. These findings suggest that ADS-J21 can be used as a new lead compound for further optimization in the development of a small-molecule fusion inhibitor.

Fully human single-domain antibody targeting a highly conserved cryptic epitope on the Nipah virus G protein.

Nipah virus infection, one of the top priority diseases recognized by the World Health Organization, underscores the urgent need to develop effective countermeasures against potential epidemics and pandemics. Here, we identify a fully human single-domain antibody that targets a highly conserved cryptic epitope situated at the dimeric interface of the Nipah virus G protein (receptor binding protein, RBP), as elucidated through structures by high-resolution cryo-electron microscopy (cryo-EM). This unique binding mode disrupts the tetramerization of the G protein, consequently obstructing the activation of the F protein and inhibiting viral membrane fusion. Furthermore, our investigations reveal that this compact antibody displays enhanced permeability across the blood-brain barrier (BBB) and demonstrates superior efficacy in eliminating pseudovirus within the brain in a murine model of Nipah virus infection, particularly compared to the well-characterized antibody m102.4 in an IgG1 format. Consequently, this single-domain antibody holds promise as a therapeutic candidate to prevent Nipah virus infections and has potential implications for vaccine development.

Development of T cell antigen-based human coronavirus vaccines against nAb-escaping SARS-CoV-2 variants.

Currently approved vaccines have been successful in preventing the severity of COVID-19 and hospitalization. These vaccines primarily induce humoral immune responses; however, highly transmissible and mutated variants, such as the Omicron variant, weaken the neutralization potential of the vaccines, thus, raising serious concerns about their efficacy. Additionally, while neutralizing antibodies (nAbs) tend to wane more rapidly than cell-mediated immunity, long-lasting T cells typically prevent severe viral illness by directly killing infected cells or aiding other immune cells. Importantly, T cells are more cross-reactive than antibodies, thus, highly mutated variants are less likely to escape lasting broadly cross-reactive T cell immunity. Therefore, T cell antigen-based human coronavirus (HCoV) vaccines with the potential to serve as a supplementary weapon to combat emerging SARS-CoV-2 variants with resistance to nAbs are urgently needed. Alternatively, T cell antigens could also be included in B cell antigen-based vaccines to strengthen vaccine efficacy. This review summarizes recent advancements in research and development of vaccines containing T cell antigens or both T and B cell antigens derived from proteins of SARS-CoV-2 variants and/or other HCoVs based on different vaccine platforms.

Discovery of a Double-Stapled Short Peptide as a Long-Acting HIV-1 Inactivator with Potential for Oral Bioavailability.

Different from most antiretroviral drugs that act as passive defenders to inhibit HIV-1 replication inside the host cell, virus inactivators can attack and inactivate HIV-1 virions without relying on their replication cycle. Herein, we describe the discovery of a hydrocarbon double-stapled helix peptide, termed D26. D26 is based on the HIV-1 gp41 protein lentiviral lytic peptide-3 motif (LLP3) sequence, which can efficiently inhibit HIV-1 infection and inactivate cell-free HIV-1 virions. It was noted that D26 was highly resistant to proteolytic degradation and exhibited a remarkably extended in vivo elimination half-life. Additionally, relative to its linear, nonstapled version, D26 exhibited much higher exposure in sanctuary sites for HIV-1. Amazingly, this lead compound also demonstrated detectable oral absorption. Thus, it can be concluded that D26 is a promising candidate for further development as a long-acting, orally applicable HIV-1 inactivator for the treatment of HIV-1 infection.

An optimized filter design approach for enhancing imaging quality in industrial linear accelerator.

BACKGROUND: The polychromatic X-rays generated by a linear accelerator (Linac) often result in noticeable hardening artifacts in images, posing a significant challenge to accurate defect identification. To address this issue, a simple yet effective approach is to introduce filters at the radiation source outlet. However, current methods are often empirical, lacking scientifically sound metrics. OBJECTIVE: This study introduces an innovative filter design method that optimizes filter performance by balancing the impact of ray intensity and energy on image quality. MATERIALS AND METHODS: Firstly, different spectra under various materials and thicknesses of filters were obtained using GEometry ANd Tracking (Geant4) simulation. Subsequently, these spectra and their corresponding incident photon counts were used as input sources to generate different reconstructed images. By comprehensively comparing the intensity differences and noise in images of defective and non-defective regions, along with considering hardening indicators, the optimal filter was determined. RESULTS: The optimized filter was applied to a Linac-based X-ray computed tomography (CT) detection system designed for identifying defects in graphite materials within high-temperature gas-cooled reactor (HTR), with defect dimensions of 2 mm. After adding the filter, the hardening effect reduced by 22%, and the Defect Contrast Index (DCI) reached 3.226. CONCLUSION: The filter designed based on the parameters of Average Difference (AD) and Defect Contrast Index (DCI) can effectively improve the quality of defect images.

Development of the IMP biosensor for rapid and stable analysis of IMP concentrations in fermentation broth.

IMP (inosinic acid) is a crucial intermediate in the purine metabolic pathway and is continuously synthesized in all cells. Besides its role as a precursor for DNA and RNA, IMP also plays a critical or essential role in cell growth, energy storage, conversion, and metabolism. In our study, we utilized the circularly permuted fluorescent protein (cpFP) and IMP dehydrogenase to screen and develop the IMP biosensor, IMPCP1. By introducing a mutation in the catalytically active site of IMPCP1, from Cys to Ala, we disrupted its ability to catalyze IMP while retaining its capability to bind to IMP without affecting the IMP concentration in the sample. To immobilize IMPCP1, we employed the SpyCatcher/SpyTag system and securely attached it to Magarose-Epoxy, resulting in the development of the IMP rapid test kit, referred to as IMPTK. The biosensor integrated into IMPTK offers enhanced stability, resistance to degradation activity, and specific recognition of IMP. It is also resistant to peroxides and temperature changes. IMPTK serves as a rapid and stable assay for analyzing IMP concentrations in fermentation broth. Within the linear range of IMP concentrations, it can be utilized as a substitute for HPLC. The IMPTK biosensor provides a reliable and efficient alternative for monitoring IMP levels, offering advantages such as speed, stability, and resistance to environmental factors.

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.

Inhibition of influenza A virus and SARS-CoV-2 infection or co-infection by griffithsin and griffithsin-based bivalent entry inhibitor.

Outbreaks of acute respiratory viral diseases, such as influenza and COVID-19 caused by influenza A virus (IAV) and SARS-CoV-2, pose a serious threat to global public health, economic security, and social stability. This calls for the development of broad-spectrum antivirals to prevent or treat infection or co-infection of IAV and SARS-CoV-2. Hemagglutinin (HA) on IAV and spike (S) protein on SARS-CoV-2, which contain various types of glycans, play crucial roles in mediating viral entry into host cells. Therefore, they are key targets for the development of carbohydrate-binding protein-based antivirals. This study demonstrated that griffithsin (GRFT) and the GRFT-based bivalent entry inhibitor GL25E (GRFT-L25-EK1) showed broad-spectrum antiviral effects against IAV infection in vitro by binding to HA in a carbohydrate-dependent manner and effectively protected mice from lethal IAV infection. Although both GRFT and GL25E could inhibit infection of SARS-CoV-2 Omicron variants, GL25E proved to be significantly more effective than GRFT and EK1 alone. Furthermore, GL25E effectively inhibited in vitro co-infection of IAV and SARS-CoV-2 and demonstrated good druggability, including favorable safety and stability profiles. These findings suggest that GL25E is a promising candidate for further development as a broad-spectrum antiviral drug for the prevention and treatment of infection or co-infection from IAV and SARS-CoV-2.IMPORTANCEInfluenza and COVID-19 are highly contagious respiratory illnesses caused by the influenza A virus (IAV) and SARS-CoV-2, respectively. IAV and SARS-CoV-2 co-infection exacerbates damage to lung tissue and leads to more severe clinical symptoms, thus calling for the development of broad-spectrum antivirals for combating IAV and SARS-CoV-2 infection or co-infection. Here we found that griffithsin (GRFT), a carbohydrate-binding protein, and GL25E, a recombinant protein consisting of GRFT, a 25 amino acid linker, and EK1, a broad-spectrum coronavirus inhibitor, could effectively inhibit IAV and SARS-CoV-2 infection and co-infection by targeting glycans on HA of IAV and spike (S) protein of SARS-CoV-2. GL25E is more effective than GRFT because GL25E can also interact with the HR1 domain in SARS-CoV-2 S protein. Furthermore, GL25E possesses favorable safety and stability profiles, suggesting that it is a promising candidate for development as a drug to prevent and treat IAV and SARS-CoV-2 infection or co-infection.

Neutralization of SARS-CoV-2 BA.2.86 and JN.1 by CF501 adjuvant-enhanced immune responses targeting the conserved epitopes in ancestral RBD.

The emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants BA.2.86 and JN.1 raise concerns regarding their potential to evade immune surveillance and spread globally. Here, we test sera from rhesus macaques immunized with 3 doses of wild-type SARS-CoV-2 receptor-binding domain (RBD)-Fc adjuvanted with the STING agonist CF501. We find that the sera can potently neutralize pseudotyped XBB.1.5, XBB.1.16, CH.1.1, EG.5, BA.2.86, and JN.1, with 50% neutralization titers ranging from 3,494 to 7,424. We also demonstrate that CF501, but not Alum, can enhance immunogenicity of the RBD from wild-type SARS-CoV-2 to improve induction of broadly neutralizing antibodies (bnAbs) with binding specificity and activity similar to those of SA55, BN03, and S309, thus exhibiting extraordinary broad-spectrum neutralizing activity. Overall, the RBD from wild-type SARS-CoV-2 also contains conservative epitopes. The RBD-Fc adjuvanted by CF501 can elicit potent bnAbs against JN.1, BA.2.86, and other XBB subvariants. This strategy can be adopted to develop broad-spectrum vaccines to combat future emerging and reemerging viral infectious diseases.

A subunit-based influenza/SARS-CoV-2 Omicron combined vaccine induced potent protective immunity in BALB/c mice.

Infection with influenza A virus (IAV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a significant risk to human life, health, and the global economy. Vaccination is one of the most effective strategies in the fight against infectious viruses. In this study, we, for the first time, have evaluated the immunogenicity and protective effect of an influenza/SARS-CoV-2 Omicron subunit combined vaccine adjuvanted with MF59 and administered to BALB/c mice. Results showed that the combined vaccine induced high levels of IgG, IgG1 , and IgG2a antibodies, as well as influenza A H1N1/California/2009 virus-specific hemagglutination-inhibiting antibodies in BALB/c mice. Moreover, this subunit combined vaccine induced high titers of neutralization antibodies against SARS-CoV-2 Omicron sublineage BA.5 pseudovirus and effectively reduced the viral load of authentic SARS-CoV-2 Omicron sublineage BA.5.2 in the cell culture supernatants. These results suggested that this subunit combined vaccine achieved protective effect against both H1N1 A/California/07/2009 strain and SARS-CoV-2 Omicron BA.5.2 variant. It is therefore expected that this study will establish the scientific foundation for the next-step development of combined vaccines against other strains or variants of IAV and SARS-CoV-2.

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.

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.

Asialoglycoprotein receptor 1 promotes SARS-CoV-2 infection of human normal hepatocytes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes multi-organ damage, which includes hepatic dysfunction, as observed in over 50% of COVID-19 patients. Angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 (ACE2) is the primary receptor for SARS-CoV-2 entry into host cells, and studies have shown the presence of intracellular virus particles in human hepatocytes that express ACE2, but at extremely low levels. Consequently, we asked if hepatocytes might express receptors other than ACE2 capable of promoting the entry of SARS-CoV-2 into cells. To address this question, we performed a genome-wide CRISPR-Cas9 activation library screening and found that Asialoglycoprotein receptor 1 (ASGR1) promoted SARS-CoV-2 pseudovirus infection of HeLa cells. In Huh-7 cells, simultaneous knockout of ACE2 and ASGR1 prevented SARS-CoV-2 pseudovirus infection. In the immortalized THLE-2 hepatocyte cell line and primary hepatic parenchymal cells, both of which barely expressed ACE2, SARS-CoV-2 pseudovirus could successfully establish an infection. However, after treatment with ASGR1 antibody or siRNA targeting ASGR1, the infection rate significantly dropped, suggesting that SARS-CoV-2 pseudovirus infects hepatic parenchymal cells mainly through an ASGR1-dependent mechanism. We confirmed that ASGR1 could interact with Spike protein, which depends on receptor binding domain (RBD) and N-terminal domain (NTD). Finally, we also used Immunohistochemistry and electron microscopy to verify that SARS-CoV-2 could infect primary hepatic parenchymal cells. After inhibiting ASGR1 in primary hepatic parenchymal cells by siRNA, the infection efficiency of the live virus decreased significantly. Collectively, these findings indicate that ASGR1 is a candidate receptor for SARS-CoV-2 that promotes infection of hepatic parenchymal cells.

SARS-CoV-2 evolution from the BA.2.86 to JN.1 variants: unexpected consequences.

SARS-CoV-2 is continuously evolving. The Omicron subvariant BA.2.86, with >30 mutations in its spike (S) protein compared with its predecessor strain BA.2, was expected to quickly become predominant worldwide, but this has not happened. Instead, its descendant strain, JN.1, with just one additional mutation, has become the predominant SARS-CoV-2 subvariant. Here, we offer a possible explanation for these unexpected consequences.

Enhanced neutralization of SARS-CoV-2 variant BA.2.86 and XBB sub-lineages by a tetravalent COVID-19 vaccine booster.

Emerging SARS-CoV-2 sub-lineages like XBB.1.5, XBB.1.16, EG.5, HK.3 (FLip), and XBB.2.3 and the variant BA.2.86 have recently been identified. Understanding the efficacy of current vaccines on these emerging variants is critical. We evaluate the serum neutralization activities of participants who received COVID-19 inactivated vaccine (CoronaVac), those who received the recently approved tetravalent protein vaccine (SCTV01E), or those who had contracted a breakthrough infection with BA.5/BF.7/XBB virus. Neutralization profiles against a broad panel of 30 sub-lineages reveal that BQ.1.1, CH.1.1, and all the XBB sub-lineages exhibit heightened resistance to neutralization compared to previous variants. However, despite their extra mutations, BA.2.86 and the emerging XBB sub-lineages do not demonstrate significantly increased resistance to neutralization over XBB.1.5. Encouragingly, the SCTV01E booster consistently induces higher neutralizing titers against all these variants than breakthrough infection does. Cellular immunity assays also show that the SCTV01E booster elicits a higher frequency of virus-specific memory B cells. Our findings support the development of multivalent vaccines to combat future variants.

Clinical development of antivirals against SARS-CoV-2 and its variants.

The unceasing global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) calls for the development of novel therapeutics. Although many newly developed antivirals and repurposed antivirals have been applied to the treatment of coronavirus disease 2019 (COVID-19), antivirals showing satisfactory clinical efficacy are few in number. In addition, the loss of sensitivity to variants of concern (VOCs) and lack of oral bioavailability have also limited the clinical application of some antivirals. These facts remind us to develop more potent and broad-spectrum antivirals with better pharmacokinetic/pharmacodynamic properties to fight against infections from SARS-CoV-2, its variants, and other human coronaviruses (HCoVs). In this review, we summarize the latest advancements in the clinical development of antivirals against infections by SARS-CoV-2 and its variants.