Single-cycle Virus Research Articles

Field-based safety evaluation of rVHSV-GΔTM in aquaculture and verification of vaccine efficacy through freeze-dried prototyping in olive flounder.

Single-cycle viruses hold great promise as fish viral vaccines due to their high protective efficacy. Although the efficacy of the vaccine in olive flounder and rainbow trout has been proven through previous research, safety must be additionally proven considering the environment of use for commercialization. This study comprehensively assesses the safety of rVHSV-GΔTM and its impact on both the host and the surrounding environment, including the coastal habitat of nearby species and seawater. We constructed an experimental tank geographically mimicking coastal conditions and evaluated its effects on co-reared olive flounder and other organisms. Results showed no detectable viral presence in co-reared organisms or concentrated culturing seawater, and the virus demonstrated limited proliferation under varying salinity conditions. The survival rate of olive flounder immunized with rVHSV-GΔTM was significantly higher (up to 90% protection against wild-type VHSV) compared to non-immunized fish. Additionally, we developed a prototype vaccine of rVHSV-GΔTM through freeze-drying. Immunization of olive flounder with the prototype vaccine conferred significantly higher protection against wild-type VHSV. These findings underscore the safety of rVHSV-GΔTM as a commercial vaccine, unaffected by the host or surrounding environment, and highlight the efficacy of freeze-drying in delivering single-cycle viruses.

Single-cycle parainfluenza virus type 5 vectors for producing recombinant proteins, including a humanized anti-V5 tag antibody.

Parainfluenza virus type 5 (PIV5) can cause either persistent or acute/lytic infections in a wide range of mammalian tissue culture cells. Here, we have generated PIV5 fusion (F)-expressing helper cell lines that support the replication of F-deleted viruses. As proof of the principle that F-deleted single-cycle infectious viruses can be used as safe and efficient expression vectors, we have cloned and expressed a humanized (Hu) version of the mouse anti-V5 tag antibody (clone SV5-Pk1). We show that multiple different cell lines can be infected and express high levels of the Hu anti-V5 antibody, with Chinese hamster ovary cells expressing 20-50 mg l-1 after 5 days when cells were grown to a density of ~1×106 cells per millilitre at the time of infection. We suggest that PIV5-based vectors may be further developed to produce recombinant proteins both in vitro and in vivo.

A BSL-2 compliant mouse model of SARS-CoV-2 infection for efficient and convenient antiviral evaluation.

Animal models of authentic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection require operation in biosafety level 3 (BSL-3) containment. In the present study, we established a mouse model employing a single-cycle infectious virus replicon particle (VRP) system of SARS-CoV-2 that can be safely handled in BSL-2 laboratories. The VRP [ΔS-VRP(G)-Luc] contains a SARS-CoV-2 genome in which the spike gene was replaced by a firefly luciferase (Fluc) reporter gene (Rep-Luci), and incorporates the vesicular stomatitis virus glycoprotein on the surface. Intranasal inoculation of ΔS-VRP(G)-Luc can successfully transduce the Rep-Luci genome into mouse lungs, initiating self-replication of Rep-Luci and, accordingly, inducing acute lung injury mimicking the authentic SARS-CoV-2 pathology. In addition, the reporter Fluc expression can be monitored using a bioluminescence imaging approach, allowing a rapid and convenient determination of viral replication in ΔS-VRP(G)-Luc-infected mouse lungs. Upon treatment with an approved anti-SARS-CoV-2 drug, VV116, the viral replication in infected mouse lungs was significantly reduced, suggesting that the animal model is feasible for antiviral evaluation. In summary, we have developed a BSL-2-compliant mouse model of SARS-CoV-2 infection, providing an advanced approach to study aspects of the viral pathogenesis, viral-host interactions, as well as the efficacy of antiviral therapeutics in the future.IMPORTANCESevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly contagious and pathogenic in humans; thus, research on authentic SARS-CoV-2 has been restricted to biosafety level 3 (BSL-3) laboratories. However, due to the scarcity of BSL-3 facilities and trained personnel, the participation of a broad scientific community in SARS-CoV-2 research had been greatly limited, hindering the advancement of our understanding on the basic virology as well as the urgently necessitated drug development. Previously, our colleagues Jin et al. had generated a SARS-CoV-2 replicon by replacing the essential spike gene in the viral genome with a Fluc reporter (Rep-Luci), which can be safely operated under BSL-2 conditions. By incorporating the Rep-Luci into viral replicon particles carrying vesicular stomatitis virus glycoprotein on their surface, and via intranasal inoculation, we successfully transduced the Rep-Luci into mouse lungs, developing a mouse model mimicking SARS-CoV-2 infection. Our model can serve as a useful platform for SARS-CoV-2 pathological studies and antiviral evaluation under BSL2 containment.

Protective efficacy against two different viral hemorrhagic septicemia virus (VHSV) isolates belonging to genotype IVa depending on various dose-boost immunizations of rVHSV-GΔTM in olive flounder

Viral vector-based vaccines are promising candidates that deliver antigens of interest into hosts. Particularly, a single-cycle virus possesses the immunogenicity of a live virus vaccine and safety of an inactivated virus vaccine because they do not produce progeny virion after infection. The rVHSV-GΔTM single-cycle, which removes the transmembrane region and C-terminal cytoplasmic region of the G gene from the KJ2008 isolate, virus demonstrates its superiority as a vaccine in controlling the viral hemorrhagic septicemia virus (VHSV) in olive flounder, although research for establishing its suitability is still lacking. Therefore, we established optimal usage through various concentrations and booster immunizations in olive flounder and confirmed the cross-protective effect against KJ2008 and GCVP-02 belonging to different subclades of the glycoprotein genotype IVa. The olive flounder were intramuscular prime or boost immunized from 1 × 102 pfu/fish to 1 × 104 pfu/fish of rVHSV-GΔTM. After four weeks of immunization, each group was challenged with KJ2008 and GCVP-02 isolates. In the prime immunization group, protective efficacy against the GCVP-02 isolate was relatively low compared with the KJ2008 isolate, but the boost immunization group showed improved protective efficacy in both isolates. Our findings suggest that the rVHSV-GΔTM single-cycle virus vaccine based on the KJ2008 isolate can be effective in controlling VHSV in the fish culture industry because it showed a protective efficacy even against other isolates and high protective efficacy upon boosting.

Single-cycle influenza virus vaccine generates lung CD8+ Trm that cross-react against viral variants and subvert virus escape mutants.

Influenza virus-specific tissue-resident memory (Trm) CD8+ T cells located along the respiratory tract provide cross-strain protection against a breadth of influenza viruses. We show that immunization with a single-cycle influenza virus vaccine candidate (S-FLU) results in the deposition of influenza virus nucleoprotein (NP)-specific CD8+ Trm along the respiratory tract that were more cross-reactive against viral variants and less likely to drive the development of cytotoxic T lymphocyte (CTL) escape mutants, as compared to the lung memory NP-specific CD8+ T cell pool established following influenza infection. This immune profile was linked to the limited inflammatory response evoked by S-FLU vaccination, which increased TCR repertoire diversity within the memory CD8+ T cell compartment. Cumulatively, this work shows that S-FLU vaccination evokes a clonally diverse, cross-reactive memory CD8+ T cell pool, which protects against severe disease without driving the virus to rapidly evolve and escape, and thus represents an attractive vaccine for use against rapidly mutating influenza viruses.

Greater Durability and Protection against Herpes Simplex Viral Disease following Immunization of Mice with Single-Cycle ΔgD-2 Compared to an Adjuvanted Glycoprotein D Protein Vaccine.

Herpes simplex viruses (HSV) cause chronic infections with significant morbidity. Prior vaccines, designed to generate neutralizing antibodies (nAbs) targeting glycoprotein D (gD), failed to provide durable protection. We adopted a different strategy and evaluated a single-cycle virus deleted in gD (ΔgD-2). ΔgD-2elicits antibodies that primarily mediate antibody-dependent cell mediated cytolysis (ADCC) and provides complete protection against clinical isolates of HSV in multiple lethal mouse models. To assess durability, we vaccinated mice (2 doses administered intramuscularly) with ΔgD-2, adjuvanted recombinant gD-2 (rgD-2/Alum-MPL), or uninfected cells as a control, and quantified antibody responses over one year. Mice (n = 5/group) were lethally challenged at 2, 4, 6, 8, and 10-months post-boost. ΔgD-2-vaccinated mice elicited a durable ADCC-mediating response, which provided complete protection against challenge at all timepoints. In contrast, rgD-2/Alum-MPL elicited only nAbs, which declined significantly within 6 months, provided only partial protection at early timepoints, and no protection after 6 months. Serum sampling after viral challenge showed that infection elicited low levels of ADCC-mediating antibodies in rgD-2/Alum-MPL-vaccinated mice and boosted the nAb response, but only after 6 months. Conversely, infection significantly and consistently boosted both the ADCC and nAbs responses in ΔgD-2-vaccinated mice. Results recapitulate clinical trial outcomes with gD vaccines, highlight the importance of ADCC, and predict that ΔgD-2 will elicit durable responses in humans.

Protection of rainbow trout (Oncorhynchus mykiss) against VHSV genotype Ia and IHNV by immunization with VHSV genotype IVa backbone-based single-cycle viruses

To evaluate the protective effect of viral hemorrhagic septicemia virus genotype IVa (VHSV IVa) genome-based single-cycle viruses against VHSV genotype Ia (VHSV Ia) and infectious hematopoietic necrosis virus (IHNV) in rainbow trout, three kinds of single-cycle VHSVs were rescued using reverse genetic technology: i) rVHSV-IaGΔTM-IVaG containing the transmembrane and cytoplasmic region-deleted G protein (GΔTM) of VHSV Ia instead of VHSV IVa full G gene ORF and having VHSV IVa G proteins on the envelope; ii) rVHSV-IaGΔTM-IaG containing VHSV Ia GΔTM instead of VHSV IVa full G gene ORF and having VHSV Ia G proteins on the envelope; iii) rVHSV-IaGΔTM-ihnvGΔTM-IVaG containing not only VHSV Ia GΔTM instead of full G gene but also IHNV GΔTM instead of NV gene and having VHSV IVa G proteins on the envelope. Rainbow trout immunized with rVHSV-IaGΔTM-IaG and rVHSV-IaGΔTM-IVaG showed significantly higher serum antibody titers against both VHSV Ia and VHSV IVa, and showed no mortality against VHSV Ia infection, while fish in the control groups showed 100% mortalities. Fish immunized with rVHSV-IaGΔTM-ihnvGΔTM-IVaG showed significantly higher serum antibody titers against VHSV IVa, VHSV Ia, and IHNV compared to fish in the control group. Immunization with rVHSV-IaGΔTM-ihnvGΔTM-IVaG induced significantly higher protection against not only VHSV Ia but also IHNV. These results suggest that the present single-cycle rVHSV-based system can be used as a platform to produce combined vaccines that can protect fish from multiple pathogenic species. However, the mechanism of the high protection against IHNV despite comparatively low antibody titer remains to be investigated.

Development of full-length cell-culture infectious clone and subgenomic replicon for a genotype 3a isolate of hepatitis C virus.

Hepatitis C virus (HCV) genotype 3 is widely distributed, and genotype 3-infected patients achieve a lower cure rate in direct-acting antiviral (DAA) therapy and are associated with a higher risk of hepatic steatosis than patients with other genotypes. Thus, the study of the virology and pathogenesis of genotype 3 HCV is increasingly relevant. Here, we developed a full-length infectious clone and a subgenomic replicon for the genotype 3a isolate, CH3a. From an infected serum, we constructed a full-length CH3a clone, however, it was nonviable in Huh7.5.1 cells. Next, we systematically adapted several intergenotypic recombinants containing Core-NS2 and 5'UTR-NS5A from CH3a, and other sequences from a replication-competent genotype 2 a clone JFH1. Adaptive mutations were identified, of which several combinations facilitated the replication of CH3a-JFH1 recombinants; however, they failed to adapt to the full-length CH3a and the recombinants containing CH3a NS5B. Thus, we attempted to separately adapt CH3a NS5B-3'UTR by constructing an intragenotypic recombinant using 5'UTR-NS5A from an infectious genotype 3a clone, DBN3acc, from which L3004P/M in NS5B and a deletion of 11 nucleotides (Δ11nt) downstream of the polyU/UC tract of the 3'UTR were identified and demonstrated to efficiently improve virus production. Finally, we combined functional 5'UTR-NS5A and NS5B-3'UTR sequences that carried the selected mutations to generate full-length CH3a with 26 or 27 substitutions (CH3acc), and both revealed efficient replication and virus spread in transfected and infected cells, releasing HCV of 104.2 f.f.u. ml-1. CH3acc was inhibited by DAAs targeting NS3/4A, NS5A and NS5B in a dose-dependent manner. The selected mutations permitted the development of subgenomic replicon CH3a-SGRep, by which L3004P, L3004M and Δ11nt were proven, together with a single-cycle virus production assay, to facilitate virus assembly, release, and RNA replication. CH3acc clones and CH3a-SGRep replicon provide new tools for the study of HCV genotype 3.

Generation of Liposomes to Study the Effect of Mycobacterium Tuberculosis Lipids on HIV-1 cis- and trans-Infections.

Tuberculosis (TB) is the leading cause of death among HIV-1-infected individuals and Mycobacterium tuberculosis (Mtb) co-infection is an early precipitate to AIDS. We aimed to determine whether Mtb strains differentially modulate cellular susceptibility to HIV-1 infection (cis- and trans-infection), via surface receptor interaction by their cell envelope lipids. Total lipids from pathogenic (lineage 4 Mtb H37Rv, CDC1551 and lineage 2 Mtb HN878, EU127) and non-pathogenic (Mycobacterium bovis BCG and Mycobacterium smegmatis) Mycobacterium strains were integrated into liposomes mimicking the lipid distribution and antigen accessibility of the mycobacterial cell wall. The resulting liposomes were tested for modulating in vitro HIV-1 cis- and trans-infection of TZM-bl cells using single-cycle infectious virus particles. Mtb glycolipids did not affect HIV-1 direct infection however, trans-infection of both R5 and X4 tropic HIV-1 strains were impaired in the presence of glycolipids from M. bovis, Mtb H37Rv and Mtb EU127 strains when using Raji-DC-SIGN cells or immature and mature dendritic cells (DCs) to capture virus. SL1, PDIM and TDM lipids were identified to be involved in DC-SIGN recognition and impairment of HIV-1 trans-infection. These findings indicate that variant strains of Mtb have differential effect on HIV-1 trans-infection with the potential to influence HIV-1 disease course in co-infected individuals.

Effect of water temperature on the protective efficacy of single-cycle rVHSV-GΔTM vaccine in olive flounder (Paralichthys olivaceus)

Water temperature is an important factor for immune responses in poikilothermic fish. Especially, it has been known that adaptive immunity is more sensitive to temperature than innate immunity in fish. The optimal temperature for olive flounder (Paralichthys olivaceus) culture is known between 20 and 25 °C, and there are several papers reporting the low or no effectiveness of inactivated vaccines in olive flounder kept at low water temperatures. Previously, we had reported that a vaccine based on single-cycle viral hemorrhagic septicemia virus (VHSV) that was modified to produce the transmembrane and C-terminal cytoplasmic region-deleted G protein in host cells (rVHSV-GΔTM) induced significantly higher survival rates in olive flounder than a vaccine of rVHSV-ΔG that had no G gene in the genome. In the present study, we evaluated the availability of rVHSV-GΔTM as a protective vaccine that can be used in olive flounder at low water temperature periods. Olive flounder fingerlings were divided into 6 groups: group 1 and 2 were kept at 14 °C, group 3 and 4 were kept at 20 °C, and group 5 and 6 were kept at 14 °C for 1 week and then shifted to 20 °C. Fish in groups 1, 3, and 5 were intramuscularly (i.m.) immunized with 8.5 × 104 PFU/fish of rVHSV-GΔTM, and fish in groups of 2, 4, and 6 were i.m. Injected with L-15 alone. In the challenge test, the survival rates of fish immunized with rVHSV-GΔTM were significantly higher than those of control group fish that were injected with L-15 alone. Among three vaccination groups (group 1, 3, and 5), group 1 showed no mortality. The cumulative mortalities of group 3 and group 5 were both 25%. While fish in control groups (group 2, 4, and 6) showed 90–100% mortalities. The qPCR genome copy number of rVHSV-GΔTM in the kidney of fish immunized at 14 °C was clearly higher than that in fish immunized at 20 °C, which suggests that higher amount of secretory viral glycoprotein would be produced in fish vaccinated at 14 °C than at 20 °C. Olive flounder immunized with rVHSV-GΔTM at 14 °C showed the serum neutralization activity as high as fish immunized at 20 °C, suggesting that the humoral immune response of olive flounder was effectively induced at lower water temperature. These results suggest that VHSV vaccines based on single-cycle viruses can be used as prophylactic vaccines even at low water temperature period.

Model of vaccine efficacy against HSV-2 superinfection of HSV-1 seropositive mice demonstrates protection by antibodies mediating cellular cytotoxicity

A majority of the world’s population is infected with HSV-1, highlighting the need for vaccines that are effective in HSV-1-seropositive hosts. We established a superinfection model by infecting mice intranasally with a sublethal dose of HSV-1, which results in high rates of seropositive, latently infected mice susceptible to HSV-2 superinfection. Sublethal HSV-1 induced a predominantly neutralizing antibody response. Vaccination of HSV-1-seropositive mice with recombinant adjuvanted glycoprotein D (rgD-2) failed to significantly boost HSV total or neutralizing antibody responses and provided no significant increased protection against HSV-2 superinfection compared to control-vaccinated HSV-1-seropositive mice. In contrast, immunization with a single-cycle virus deleted in gD (ΔgD-2) significantly boosted total HSV-specific antibody titers and elicited new antibody-dependent cell-mediated cytotoxicity responses, providing complete protection from death following HSV-2 superinfection. This model recapitulates clinical responses to natural infection and the rgD-2 vaccine trial outcomes and suggests that ΔgD-2 may prove protective in HSV-1-seropositive hosts.

Enhanced production of infectious particles by adaptive modulation of C-prM processing and C-C interaction during propagation of dengue pseudoinfectious virus in stable CprME-expressing cells.

Dengue virus assembly involves the encapsidation of genomic RNA by the capsid protein (C) and the acquisition of an envelope comprising the premembrane (prM) and envelope (E) glycoproteins. This rapid process, lacking in detectable nucleocapsid intermediates, may impose authentic C-prM-E arrangement as a prerequisite for efficient particle assembly. A mosquito cell-based complementation system was employed in this study to investigate the possibility that expression of the three structural proteins in trans allows the efficient production of a partially C-deleted dengue virus as compared to the presence of C alone. Following the transfection of ΔC56-capped RNA transcripts into C6/36 cells transiently expressing C or CprME, the production of the single-cycle virus was comparable. Subsequent propagation in the stable CprME-expressing clone, however, supported virus adaptation leading to acquisition of the L29P and S101F (PF) dual mutations in the C protein. The triple mutant, ΔC56(PF), exhibited enhanced levels of virus replication, specific infectivity and frequent increases of intracellular C dimer, as compared with ΔC56 in the CprME-clone. The PF mutations were associated with the accumulation of truncated CprM in ΔC56(PF)-infected cells, and uncleaved CprM as well as reduced intracellular C-dimer when the dual mutations were introduced into the wild-type dengue virus genetic background. These results indicate that the PF mutations may exert a replication-enhancing effect for the triple mutant virus by relieving the interference of trans-complementing structural proteins during viral assembly and suggest that the C-prM-E arrangement may be advantageous for pseudoinfectious virus production.

A Single-Cycle Adenovirus Type 7 Vaccine for Prevention of Acute Respiratory Disease.

Adenovirus type 7 (Ad7) infection is associated with acute respiratory disease (ARD), especially in military recruits living in close quarters. Recently, several outbreaks of Ad7 infections have occurred in civilian populations, with some cases leading to death. However, the current Ad7 vaccine is licensed for use only in military recruits because it utilizes an orally delivered wild type virus which is shed in the stool for 28 days after immunization. This poses a safety risk due to the possibility of virus spread to vulnerable populations. To address the need for a safer Ad7 vaccine for use in civilian populations, we developed a single-cycle Ad7 virus (scAd7). This scAd7 virus is deleted for the Ad7 fiber protein, so that viruses produced outside of complementing cells lines lack this essential structural protein and have severely reduced infectivity. In vitro studies in noncomplementing A549 cells showed that the scAd7 virus has genomic DNA replication kinetics and Ad7 hexon expression similar to a replication-competent virus; however, virus progeny produced after infection has impaired infectivity. Therefore, this scAd7 virus combines the safety advantages of a replication-defective virus with the increased Ad7 gene expression of a replication-competent virus. Due to these advantages, we believe that scAd7 viruses should be further studied as an alternative, safer Adenovirus 7 vaccine.

Effect of CXCL12-expressing viral hemorrhagic septicemia virus replicon particles on leukocytes migration and vaccine efficacy in olive flounder (Paralichthys olivaceus)

Viral replicon particles are single-cycle viruses defective for function(s) needed for viral replication, which allow them to be recognized as a safer form for the vaccination of animals compared to attenuated live viruses. However, deletion of genes that are critical for the induction of protective immunity can diminish the vaccine potential of viral replicon particles. Therefore, the manipulation of viral replicon particles to produce a molecular adjuvant can be a way to increase immunogenicity of vaccines based on viral replicon particles. Chemokines are a class of chemotactic cytokines that control the migration of diverse cells of vertebrates. CXC chemokine ligand 12 (CXCL12) binds to a receptor CXCR4, and CXCL12-CXCR4 signaling plays an important role in the migration of hematopoietic cells during embryogenesis and the attraction of leukocytes. In the present study, to evaluate the possible use of CXCL12 as a molecular adjuvant for an rVHSV-ΔG vaccine and to know differences between CXCL12a and CXCL12b in the adjuvant ability, we rescued VHSV replicon particles that are expressing olive flounder CXCL12a, CXCL12b, or eGFP (rVHSV-ΔG-CXCL12a, rVHSV-ΔG-CXCL12b, or rVHSV-ΔG-eGFP), and compared the ability to attract olive flounder leucocytes and to induce protection against a VHSV challenge. In the leukocytes migration assay, supernatants collected from cells infected with rVHSV-ΔG-CXCL12a and rVHSV-ΔG-CXCL12b showed significantly higher ability to attract olive flounder leukocytes than the supernatant of cells infected with rVHSV-ΔG-eGFP. Moreover, the significantly higher number of leukocytes were attracted to rVHSV-CXCL12a supernatant compared to rVHSV-CXCL12b supernatant, suggesting that CXCL12a would be more appropriate for the induction of immunity than CXCL12b in olive flounder. In the immunization experiment, olive flounder immunized with rVHSV-ΔG-CXCL12a showed significantly higher survival rate than fish immunized with rVHSV-ΔG-CXCL12b or rVHSV-ΔG-eGFP. In addition, fish immunized with rVHSV-ΔG-CXCL12a showed the highest serum neutralization activity. These results suggest the availability of CXCL12a for a molecular adjuvant of vaccines based on VHSV replicon particles.

Vitamin D derivatives inhibit hepatitis C virus production through the suppression of apolipoprotein

Supplementation with vitamin D (VD) has been reported to improve the efficacy of interferon-based therapy for chronic hepatitis C. We found that 25-hydroxyvitamin D3 (25-(OH)D3), one of the metabolites of VD, has antiviral effects by inhibiting the infectious virus production of the hepatitis C virus (HCV). In this study, to clarify the underlying mechanisms of the anti-HCV effects, we searched VD derivatives that have anti-HCV effects and identified the common target molecule in the HCV life cycle by using an HCV cell culture system. After infection of Huh-7.5.1 cells with cell culture-generated HCV, VD derivatives were added to culture media, and the propagation of HCV was assessed by measuring the HCV core antigen levels in culture media and cell lysates. To determine the step in the HCV life cycle affected by these compounds, the single-cycle virus production assay was used with a CD81-negative cell line. Of the 14 structural derivatives of VD, an anti-HCV effect was detected in 9 compounds. Cell viability was not affected by these effective compounds. The 2 representative VD derivatives inhibited the infectious virus production in the single-cycle virus production assay. Treatment with these compounds and 25-(OH)D3 suppressed the expression of apolipoprotein A1 and C3, which are known to be involved in infectious virus production of HCV, and the knockdown of these apolipoproteins reduced infectious virus production. In conclusion, we identified several compounds with anti-HCV activity by screening VD derivatives. These compounds reduce the infectious virus production of HCV by suppressing the expression of apolipoproteins in host cells.

Distinct antiviral signatures revealed by the magnitude and round of influenza virus replication in vivo

Influenza virus has a broad cellular tropism in the respiratory tract. Infected epithelial cells sense the infection and initiate an antiviral response. To define the antiviral response at the earliest stages of infection we used a series of single-cycle reporter viruses. These viral probes demonstrated cells in vivo harbor a range in magnitude of virus replication. Transcriptional profiling of cells supporting different levels of replication revealed tiers of IFN-stimulated gene expression. Uninfected cells and cells with blunted replication expressed a distinct and potentially protective antiviral signature, while cells with high replication expressed a unique reserve set of antiviral genes. Finally, we used these single-cycle reporter viruses to determine the antiviral landscape during virus spread, which unveiled disparate protection of epithelial cell subsets mediated by IFN in vivo. Together these results highlight the complexity of virus-host interactions within the infected lung and suggest that magnitude and round of replication tune the antiviral response.

Characterization of Influenza Virus Pseudotyped with Ebolavirus Glycoprotein.

ABSTRACTWe have produced a new Ebola virus pseudotype, E-S-FLU, that can be handled in biosafety level 1/2 containment for laboratory analysis. The E-S-FLU virus is a single-cycle influenza virus coated with Ebolavirus glycoprotein, and it encodes enhanced green fluorescence protein as a reporter that replaces the influenza virus hemagglutinin. MDCK-SIAT1 cells were transduced to express Ebolavirus glycoprotein as a stable transmembrane protein for E-S-FLU virus production. Infection of cells with the E-S-FLU virus was dependent on the Niemann-Pick C1 protein, which is the well-characterized receptor for Ebola virus entry at the late endosome/lysosome membrane. The E-S-FLU virus was neutralized specifically by an anti-Ebolavirus glycoprotein antibody and a variety of small drug molecules that are known to inhibit the entry of wild-type Ebola virus. To demonstrate the application of this new Ebola virus pseudotype, we show that a single laboratory batch was sufficient to screen a library (LOPAC1280; Sigma) of 1,280 pharmacologically active compounds for inhibition of virus entry. A total of 215 compounds inhibited E-S-FLU virus infection, while only 22 inhibited the control H5-S-FLU virus coated in H5 hemagglutinin. These inhibitory compounds have very dispersed targets and mechanisms of action, e.g., calcium channel blockers, estrogen receptor antagonists, antihistamines, serotonin uptake inhibitors, etc., and this correlates with inhibitor screening results obtained with other pseudotypes or wild-type Ebola virus in the literature. The E-S-FLU virus is a new tool for Ebola virus cell entry studies and is easily applied to high-throughput screening assays for small-molecule inhibitors or antibodies.IMPORTANCE Ebola virus is in the Filoviridae family and is a biosafety level 4 pathogen. There are no FDA-approved therapeutics for Ebola virus. These characteristics warrant the development of surrogates for Ebola virus that can be handled in more convenient laboratory containment to study the biology of the virus and screen for inhibitors. Here we characterized a new surrogate, named E-S-FLU virus, that is based on a disabled influenza virus core coated with the Ebola virus surface protein but does not contain any genetic information from the Ebola virus itself. We show that E-S-FLU virus uses the same cell entry pathway as wild-type Ebola virus. As an example of the ease of use of E-S-FLU virus in biosafety level 1/2 containment, we showed that a single production batch could provide enough surrogate virus to screen a standard small-molecule library of 1,280 candidates for inhibitors of viral entry.

Vector Order Determines Protection against Pathogenic Simian Immunodeficiency Virus Infection in a Triple-Component Vaccine by Balancing CD4+ and CD8+ T-Cell Responses.

An effective AIDS vaccine should elicit strong humoral and cellular immune responses while maintaining low levels of CD4+ T-cell activation to avoid the generation of target cells for viral infection. The present study investigated two prime-boost regimens, both starting vaccination with single-cycle immunodeficiency virus, followed by two mucosal boosts with either recombinant adenovirus (rAd) or fowlpox virus (rFWPV) expressing SIVmac239 or SIVmac251 gag/pol and env genes, respectively. Finally, vectors were switched and systemically administered to the reciprocal group of animals. Only mucosal rFWPV immunizations followed by systemic rAd boost significantly protected animals against a repeated low-dose intrarectal challenge with pathogenic SIVmac251, resulting in a vaccine efficacy (i.e., risk reduction per exposure) of 68%. Delayed viral acquisition was associated with higher levels of activated CD8+ T cells and Gag-specific gamma interferon (IFN-γ)-secreting CD8+ cells, low virus-specific CD4+ T-cell responses, and low Env antibody titers. In contrast, the systemic rFWPV boost induced strong virus-specific CD4+ T-cell activity. rAd and rFWPV also induced differential patterns of the innate immune responses, thereby possibly shaping the specific immunity. Plasma CXCL10 levels after final immunization correlated directly with virus-specific CD4+ T-cell responses and inversely with the number of exposures to infection. Also, the percentage of activated CD69+ CD8+ T cells correlated with the number of exposures to infection. Differential stimulation of the immune response likely provided the basis for the diverging levels of protection afforded by the vaccine regimen.IMPORTANCE A failed phase II AIDS vaccine trial led to the hypothesis that CD4+ T-cell activation can abrogate any potentially protective effects delivered by vaccination or promote acquisition of the virus because CD4+ T helper cells, required for an effective immune response, also represent the target cells for viral infection. We compared two vaccination protocols that elicited similar levels of Gag-specific immune responses in rhesus macaques. Only the animal group that had a low level of virus-specific CD4+ T cells in combination with high levels of activated CD8+ T cells was significantly protected from infection. Notably, protection was achieved despite the lack of appreciable Env antibody titers. Moreover, we show that both the vector and the route of immunization affected the level of CD4+ T-cell responses. Thus, mucosal immunization with FWPV-based vaccines should be considered a potent prime in prime-boost vaccination protocols.

Maternal Immunization with a Single-Cycle Herpes Simplex Virus (HSV) Candidate Vaccine, ΔgD-2, Protects Neonatal Mice from Lethal Viral Challenge

BackgroundPerinatal HSV is associated with ~60% mortality if untreated and with substantial morbidity even with appropriate therapy. We recently engineered a single-cycle virus deleted in glycoprotein-D (ΔgD-2) that induces high-titer antibodies (Abs) that are non-neutralizing but activate the Fc receptor (FcR) to elicit antibody-dependent cellular cytotoxicity (ADCC). Immunization with ΔgD-2 completely protects adult mice from HSV-1 and HSV-2 disease following vaginal, skin, intraocular, or intranasal challenge and prevents the establishment of latency (ELife, 2014, JCI Insight, 2016). Thus we hypothesize that maternal immunization with ΔgD-2 and/or passive transfer of immune serum will protect neonates from HSV.MethodsFour- to 6-week-old C57Bl/6 female mice were primed and boosted at 3-week intervals with ΔgD-2 or an equal volume of uninfected cell lysates (VD60 cells). Two weeks post-boost, mice were mated and pups were challenged with a lethal dose of HSV-1 (Bx31.1) at day 7 of birth. To differentiate the contribution of transplacental vs. colostrum Abs, mothers were switched at birth. Alternatively, 7-day-old mice born to nonimmunized mothers received a single dose of immune serum (400 μg total Ab) intraperitoneally at time of intranasal challenge.ResultsThirty-eight of 47 (81%) of the pups born to and nursed by ΔgD-2-immunized mothers survived, exhibited little or no signs of disease and were protected from latency as measured by quantifying HSV DNA by PCR in neuronal tissue. In contrast, 12/14 (86%) of pups born to control vaccinated and nursed mice developed neurological signs of disease and died (P < 0.0001, Fisher’s exact test). Survival was associated with increased ADCC Abs in the serum of neonatal mice. In contrast, passive transfer of immune serum, which consistently protects adult mice from infection, did not protect neonates. If newborns born to immunized mice suckled with control mice, protection was partially abrogated (11/19, 58% survival), suggesting that both systemic and mucosal Abs are required for complete protection.ConclusionMaternal vaccination with ΔgD-2 provides significant protection against intranasal neonatal challenge but may require exposure to systemic and mucosal Abs.Disclosures W. R. Jacobs Jr., xvax: Scientific Advisor, Research support; B. C. Herold, X-vax: Grant Investigator, Research grant and Research support

Antiviral Activity of Dual-acting Hydrocarbon-stapled Peptides against HIV-1 Predominantly Circulating in China

ObjectiveNew rationally designed i,i+7-hydrocarbon-stapled peptides that target both HIV-1 assembly and entry have been shown to have antiviral activity against HIV-1 subtypes circulating in Europe and North America. Here, we aimed to evaluate the antiviral activity of these peptides against HIV-1 subtypes predominantly circulating in China. MethodsThe antiviral activity of three i,i+7-hydrocarbon-stapled peptides, NYAD-36, NYAD-67, and NYAD-66, against primary HIV-1 CRF07_BC and CRF01_AE isolates was evaluated in peripheral blood mononuclear cells (PBMCs). The activity against the CRF07_BC and CRF01_AE Env-pseudotyped viruses was analyzed in TZM-bl cells. ResultsWe found that all the stapled peptides were effective in inhibiting infection by all the primary HIV-1 isolates tested, with 50% inhibitory concentration toward viral replication (IC50) in the low micromolar range. NYAD-36 and NYAD-67 showed better antiviral activity than NYAD-66 did. We further evaluated the sensitivity of CRF01_AE and CRF07_BC Env-pseudotyped viruses to these stapled peptides in a single-cycle virus infectivity assay. As observed with the primary isolates, the IC50s were in the low micromolar range, and NYAD-66 was less effective than NYAD-36 and NYAD-67. ConclusionHydrocarbon-stapled peptides appear to have broad antiviral activity against the predominant HIV-1 viruses in China. This finding may provide the impetus to the rational design of peptides for future antiviral therapy.