Safe and effective two-in-one replicon-and-VLP minispike vaccine for COVID-19: Protection of mice after a single immunization.

Vaccines of outstanding efficiency, safety, and public acceptance are needed to halt the current SARS-CoV-2 pandemic. Concerns include potential side effects caused by the antigen itself and safety of viral DNA and RNA delivery vectors. The large SARS-CoV-2 spike (S) protein is the main target of current COVID-19 vaccine candidates but can induce non-neutralizing antibodies, which might cause vaccination-induced complications or enhancement of COVID-19 disease. Besides, encoding of a functional S in replication-competent virus vector vaccines may result in the emergence of viruses with altered or expanded tropism. Here, we have developed a safe single round rhabdovirus replicon vaccine platform for enhanced presentation of the S receptor-binding domain (RBD). Structure-guided design was employed to build a chimeric minispike comprising the globular RBD linked to a transmembrane stem-anchor sequence derived from rabies virus (RABV) glycoprotein (G). Vesicular stomatitis virus (VSV) and RABV replicons encoding the minispike not only allowed expression of the antigen at the cell surface but also incorporation into the envelope of secreted non-infectious particles, thus combining classic vector-driven antigen expression and particulate virus-like particle (VLP) presentation. A single dose of a prototype replicon vaccine complemented with VSV G, VSVΔG-minispike-eGFP (G), stimulated high titers of SARS-CoV-2 neutralizing antibodies in mice, equivalent to those found in COVID-19 patients, and protected transgenic K18-hACE2 mice from COVID-19-like disease. Homologous boost immunization further enhanced virus neutralizing activity. The results demonstrate that non-spreading rhabdovirus RNA replicons expressing minispike proteins represent effective and safe alternatives to vaccination approaches using replication-competent viruses and/or the entire S antigen.

Defective Monocyte Enzymatic Function and an Inhibitory Immune Phenotype in Human Immunodeficiency Virus-Exposed Uninfected African Infants in the Era of Antiretroviral Therapy.

BACKGROUND: Human immunodeficiency virus-exposed uninfected (HEU) infants are a rapidly expanding population in sub-Saharan Africa and are highly susceptible to encapsulated bacterial disease in the first year of life. The mechanism of this increased risk is still poorly understood. We investigated whether human immunodeficiency virus (HIV)-exposure dysregulates HEU immunity, vaccine-antibody production, and human herpes virus amplify this effect. METHODS: Thirty-four HIV-infected and 44 HIV-uninfected pregnant women were recruited into the birth cohort and observed up to 6 weeks of age; and then a subsequent 43 HIV-infected and 61 HIV-uninfected mother-infant pairs were recruited into a longitudinal infant cohort at either: 5-7 to 14-15; or 14-15 to 18-23 weeks of age. We compared monocyte function, innate and adaptive immune cell phenotype, and vaccine-induced antibody responses between HEU and HIV-unexposed uninfected (HU) infants. RESULTS: We demonstrate (1) altered monocyte phagosomal function and B-cell subset homeostasis and (2) lower vaccine-induced anti-Haemophilus influenzae type b (Hib) and anti-tetanus toxoid immunoglobulin G titers in HEU compared with HU infants. Human herpes virus infection was similar between HEU and HU infants. CONCLUSIONS: In the era of antiretroviral therapy-mediated viral suppression, HIV exposure may dysregulate monocyte and B-cell function, during the vulnerable period of immune maturation. This may contribute to the high rates of invasive bacterial disease and pneumonia in HEU infants.

Labyrinthopeptins Exert Broad-Spectrum Antiviral Activity through Lipid-Binding-Mediated Virolysis.

To counteract the serious health threat posed by known and novel viral pathogens, drugs that target a variety of viruses through a common mechanism have attracted recent attention due to their potential in treating (re)emerging infections, for which direct-acting antivirals are not available. We found that labyrinthopeptins A1 and A2, the prototype congeners of carbacyclic lanthipeptides, inhibit the proliferation of diverse enveloped viruses, including dengue virus, Zika virus, West Nile virus, hepatitis C virus, chikungunya virus, Kaposi's sarcoma-associated herpesvirus, cytomegalovirus, and herpes simplex virus, in the low micromolar to nanomolar range. Mechanistic studies on viral particles revealed that labyrinthopeptins induce a virolytic effect through binding to the viral membrane lipid phosphatidylethanolamine (PE). These effects are enhanced by a combined equimolar application of both labyrinthopeptins, and a clear synergism was observed across a concentration range corresponding to 10% to 90% inhibitory concentrations of the compounds. Time-resolved experiments with large unilamellar vesicles (LUVs) reveal that membrane lipid raft compositions (phosphatidylcholine [PC]/PE/cholesterol/sphingomyelin at 17:10:33:40) are particularly sensitive to labyrinthopeptins in comparison to PC/PE (90:10) LUVs, even though the overall PE amount remains constant. Labyrinthopeptins exhibited low cytotoxicity and had favorable pharmacokinetic properties in mice (half-life [t1/2] = 10.0 h), which designates them promising antiviral compounds acting by an unusual viral lipid targeting mechanism.IMPORTANCE For many viral infections, current treatment options are insufficient. Because the development of each antiviral drug is time-consuming and expensive, the prospect of finding broad-spectrum antivirals that can fight multiple, diverse viruses-well-known viruses as well as (re)emerging species-has gained attention, especially for the treatment of viral coinfections. While most known broad-spectrum agents address processes in the host cell, we found that targeting lipids of the free virus outside the host cell with the natural products labyrinthopeptin A1 and A2 is a viable strategy to inhibit the proliferation of a broad range of viruses from different families, including chikungunya virus, dengue virus, Zika virus, Kaposi's sarcoma-associated herpesvirus, and cytomegalovirus. Labyrinthopeptins bind to viral phosphatidylethanolamine and induce virolysis without exerting cytotoxicity on host cells. This represents a novel and unusual mechanism to tackle medically relevant viral infections.

Structural and phenotypic analysis of Chikungunya virus RNA replication elements.

Chikungunya virus (CHIKV) is a re-emerging, pathogenic Alphavirus transmitted to humans by Aedes spp. mosquitoes. We have mapped the RNA structure of the 5' region of the CHIKV genome using selective 2'-hydroxyl acylation analysed by primer extension (SHAPE) to investigate intramolecular base-pairing at single-nucleotide resolution. Taking a structure-led reverse genetic approach, in both infectious virus and sub-genomic replicon systems, we identified six RNA replication elements essential to efficient CHIKV genome replication - including novel elements, either not previously analysed in other alphaviruses or specific to CHIKV. Importantly, through a reverse genetic approach we demonstrate that the replication elements function within the positive-strand genomic copy of the virus genome, in predominantly structure-dependent mechanisms during efficient replication of the CHIKV genome. Comparative analysis in human and mosquito-derived cell lines reveal that a novel element within the 5'UTR is essential for efficient replication in both host systems, while those in the adjacent nsP1 encoding region are specific to either vertebrate or invertebrate host cells. In addition to furthering our knowledge of fundamental aspects of the molecular virology of this important human pathogen, we foresee that results from this study will be important for rational design of a genetically stable attenuated vaccine.

A central hydrophobic E1 region controls the pH range of hepatitis C virus membrane fusion and susceptibility to fusion inhibitors.

BACKGROUND & AIMS: Hepatitis C virus (HCV) infection causes chronic liver disease. Antivirals have been developed and cure infection. However, resistance can emerge and salvage therapies with alternative modes of action could be useful. Several licensed drugs have emerged as HCV entry inhibitors and are thus candidates for drug repurposing. We aimed to dissect their mode of action, identify improved derivatives and determine their viral targets. METHODS: HCV entry inhibition was tested for a panel of structurally related compounds, using chimeric viruses representing diverse genotypes, in addition to viruses containing previously determined resistance mutations. Chemical modeling and synthesis identified improved derivatives, while generation of susceptible and non-susceptible chimeric viruses pinpointed E1 determinants of compound sensitivity. RESULTS: Molecules of the diphenylpiperazine, diphenylpiperidine, phenothiazine, thioxanthene, and cycloheptenepiperidine chemotypes inhibit HCV infection by interfering with membrane fusion. These molecules and a novel p-methoxy-flunarizine derivative with improved efficacy preferentially inhibit genotype 2 viral strains. Viral residues within a central hydrophobic region of E1 (residues 290-312) control susceptibility. At the same time, viral features in this region also govern pH-dependence of viral membrane fusion. CONCLUSIONS: Small molecules from different chemotypes related to flunarizine preferentially inhibit HCV genotype 2 membrane fusion. A hydrophobic region proximal to the putative fusion loop controls sensitivity to these drugs and the pH range of membrane fusion. An algorithm considering viral features in this region predicts viral sensitivity to membrane fusion inhibitors. Resistance to flunarizine correlates with more relaxed pH requirements for fusion. LAY SUMMARY: This study describes diverse compounds that act as HCV membrane fusion inhibitors. It defines viral properties that determine sensitivity to these molecules and thus provides information to identify patients that may benefit from treatment with membrane fusion inhibitors.

Hepacivirus NS3/4A Proteases Interfere with MAVS Signaling in both Their Cognate Animal Hosts and Humans: Implications for Zoonotic Transmission.

Multiple novel members of the genus Hepacivirus have recently been discovered in diverse mammalian species. However, to date, their replication mechanisms and zoonotic potential have not been explored in detail. The NS3/4A serine protease of hepatitis C virus (HCV) is critical for cleavage of the viral polyprotein. It also cleaves the cellular innate immune adaptor MAVS, thus decreasing interferon (IFN) production and contributing to HCV persistence in the human host. To investigate the conservation of fundamental aspects of the hepaciviral life cycle, we explored if MAVS cleavage and suppression of innate immune signaling represent a common mechanism employed across different clades of the genus Hepacivirus to enhance viral replication. To estimate the zoonotic potential of these nonhuman hepaciviruses, we assessed if their NS3/4A proteases were capable of cleaving human MAVS. NS3/4A proteases of viruses infecting colobus monkeys, rodents, horses, and cows cleaved the MAVS proteins of their cognate hosts and interfered with the ability of MAVS to induce the IFN-ß promoter. All NS3/4A proteases from nonhuman viruses readily cleaved human MAVS. Thus, NS3/4A-dependent cleavage of MAVS is a conserved replication strategy across multiple clades within the genus Hepacivirus Human MAVS is susceptible to cleavage by these nonhuman viral proteases, indicating that it does not pose a barrier for zoonotic transmission of these viruses to humans. IMPORTANCE: Virus infection is recognized by cellular sensor proteins triggering innate immune signaling and antiviral defenses. While viruses have evolved strategies to thwart these antiviral programs in their cognate host species, these evasion mechanisms are often ineffective in a novel host, thus limiting viral transmission across species. HCV, the best-characterized member of the genus Hepacivirus within the family Flaviviridae, uses its NS3/4A protease to disrupt innate immune signaling by cleaving the cellular adaptor protein MAVS. Recently, a large number of HCV-related viruses have been discovered in various animal species, including wild, livestock, and companion animals. We show that the NS3/4A proteases of these hepaciviruses from different animals and representing various clades of the genus cleave their cognate host MAVS proteins in addition to human MAVS. Therefore, cleavage of MAVS is a common strategy of hepaciviruses, and human MAVS is likely unable to limit replication of these nonhuman viruses upon zoonotic exposure.

Asymptomatic HIV-infected individuals on antiretroviral therapy exhibit impaired lung CD4(+) T-cell responses to mycobacteria.

RATIONALE: HIV-infected persons on antiretroviral therapy (ART) remain at higher risk of pulmonary tuberculosis (TB) than HIV-uninfected individuals. This increased susceptibility may be caused by impairment of alveolar macrophage (AM) function and/or mycobacteria-specific alveolar CD4(+) T-cell responses observed in HIV-infected ART-naive adults. OBJECTIVES: To determine whether ART was associated with improvement in both AM function, assessed by phagosomal proteolysis, and alveolar CD4(+) T-cell responses to Mycobacterium in HIV-infected individuals. METHODS: Peripheral blood was drawn and bronchoalveolar lavage (BAL) performed on healthy, 35 HIV-uninfected, 25 HIV-infected ART-naive, and 50 HIV-infected ART-treated asymptomatic adults. Phagosomal proteolysis of AM was assessed with fluorogenic beads. Mycobacteria-specific CD4(+) T-cell responses were measured by intracellular cytokine staining. MEASUREMENTS AND MAIN RESULTS: HIV-infected adults on ART exhibited lower plasma HIV viral load and higher blood CD4(+) T-cell count than ART-naive adults. AM proteolysis and total mycobacteria-specific Th1 CD4(+) T-cell responses in individuals on ART for greater than or equal to 4 years were similar to HIV-uninfected control subjects but those on ART for less than 4 years had impaired responses. Total influenza-specific alveolar Th1 CD4(+) T-cell responses were intact in all individuals receiving ART. In contrast, BAL and blood mycobacteria-specific polyfunctional CD4(+) T-cell responses were impaired in adults on ART irrespective of duration. CONCLUSIONS: AM and mycobacteria-specific alveolar CD4(+) T-cell responses in HIV-infected adults on ART for less than 4 years are impaired and may partly explain the high risk of TB in HIV-infected individuals on ART. Strategies to augment ART to improve lung immune cell function and reduce the high incidence of TB in HIV-infected adults who initiate ART should be investigated.

Animal Models for Hepatitis E virus.

Hepatitis E virus (HEV) is an underdiagnosed pathogen with approximately 20 million infections each year and currently the most common cause of acute viral hepatitis. HEV was long considered to be confined to developing countries but there is increasing evidence that it is also a medical problem in the Western world. HEV that infects humans belongs to the Orthohepevirus A species of the Hepeviridae family. Novel HEV-like viruses have been observed in a variety of animals and some have been shown to be able to cross the species barrier, causing infection in humans. Several cell culture models for HEV have been established in the past years, but their efficiency is usually relatively low. With the circulation of this virus and related viruses in a variety of species, several different animal models have been developed. In this review, we give an overview of these animal models, indicate their main characteristics, and highlight how they may contribute to our understanding of the basic aspects of the viral life cycle and cross-species infection, the study of pathogenesis, and the evaluation of novel preventative and therapeutic strategies.

Alveolar T-helper 17 responses to streptococcus pneumoniae are preserved in ART-untreated and treated HIV-infected Malawian adults.

OBJECTIVE: We explored if HIV infection is associated with impaired T-Helper 17 responses against Streptococcus pneumoniae in the lung. METHODS: We recruited 30 HIV-uninfected healthy controls, 23 asymptomatic HIV-infected adults not on ART, and 40 asymptomatic HIV-infected adults on ART (Median time 3.5yrs), in whom we collected bronchoalveolar lavage fluid. We measured alveolar CD4+ T cell immune responses following stimulation with pneumococcal cell culture supernatant using flow cytometry-based intracellular cytokine staining. RESULTS: We found that the proportion of alveolar CD4+ T cells producing IL-17A following stimulation with pneumococcal cell culture supernatant (CCS) was similar between HIV-uninfected controls and ART-naïve HIV-infected adults (0.10% vs. 0.14%; p = 0.9273). In contrast, the proportion and relative absolute counts of CD4+ T cells producing IL-17A in response to pneumococcal CCS were higher in ART-treated HIV-infected adults compared HIV-uninfected controls (0.22% vs. 0.10%, p = 0.0166; 5420 vs. 1902 cells/100 ml BAL fluid; p = 0.0519). The increase in relative absolute numbers of IL-17A-producing alveolar CD4+ T cells in ART-treated individuals was not correlated with the peripheral blood CD4+ T cell count (r=-0.1876, p = 0.1785). CONCLUSION: Alveolar Th17 responses against S. pneumoniae are preserved in HIV-infected adults. This suggests that there are other alternative mechanisms that are altered in HIV-infected individuals that render them more susceptible to pneumococcal pneumonia.

Synthetic Polymer with a Structure-Driven Hepatic Deposition and Curative Pharmacological Activity in Hepatic Cells.

Synthetic polymers make strong contributions as tools for delivery of biological drugs and chemotherapeutics. The most praised characteristic of polymers in these applications is complete lack of pharmacological function such as to minimize the side effects within the human body. In contrast, synthetic polymers with curative pharmacological activity are truly rare. Moreover, such activity is typically nonspecific rather than structure-defined. In this work, we present the discovery of poly(ethylacrylic acid) (PEAA) as a polymer with a suit of structure-defined, unexpected, pharmacological, and pharmacokinetic properties not observed in close structural analogues. Specifically, PEAA reveals capacity to bind to albumin with ensuing natural hepatic deposition in vivo and exhibits concurrent inhibitory activity against the hepatitis C virus and inflammation in hepatic cells. Our findings provide a view on synthetic polymers as curative, functional agents and present PEAA as a unique biomedical tool with applications related to health of the human liver.

HIV-associated disruption of lung cytokine networks is incompletely restored in asymptomatic HIV-infected Malawian adults on antiretroviral therapy.

Disruption of lung cytokine networks during chronic HIV infection is incompletely restored in individuals on antiretroviral therapy.