Antiviral Activity of Influenza A Virus Defective Interfering Particles against SARS-CoV-2 Replication In Vitro through Stimulation of Innate Immunity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) emerged in late 2019 and resulted in a devastating pandemic. Although the first approved vaccines were already administered by the end of 2020, worldwide vaccine availability is still limited. Moreover, immune escape variants of the virus are emerging against which the current vaccines may confer only limited protection. Further, existing antivirals and treatment options against COVID-19 show only limited efficacy. Influenza A virus (IAV) defective interfering particles (DIPs) were previously proposed not only for antiviral treatment of the influenza disease but also for pan-specific treatment of interferon (IFN)-sensitive respiratory virus infections. To investigate the applicability of IAV DIPs as an antiviral for the treatment of COVID-19, we conducted in vitro co-infection experiments with cell culture-derived DIPs and the IFN-sensitive SARS-CoV-2 in human lung cells. We show that treatment with IAV DIPs leads to complete abrogation of SARS-CoV-2 replication. Moreover, this inhibitory effect was dependent on janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling. Further, our results suggest boosting of IFN-induced antiviral activity by IAV DIPs as a major contributor in suppressing SARS-CoV-2 replication. Thus, we propose IAV DIPs as an effective antiviral agent for treatment of COVID-19, and potentially also for suppressing the replication of new variants of SARS-CoV-2.

Safety, Immunogenicity, and Effectiveness of Defective Viral Particles Arising in Mast Cells Against Influenza in Mice.

Mast cells play pivotal roles in the pathogenesis of influenza A virus (IAV) infections. Defective viral particles (DPs) often arise during IAV replication, which can interfere with the replication of infectious viruses and stimulate the antiviral response of host cells. Therefore, DPs are expected to have immune-protective functions in clinic. However, the potent immunogenicity and effectiveness of DPs arising in mast cells during IAV replication have not been reported. In the present study, we showed that DPs generated in the human mastocytoma cell line HMC-1 following H1N1 infection were safe to mice after vaccination. Compared with lung adenocarcinoma cells, A549, DPs generated in infected mast cells had much better immunostimulatory activity, enhancing both humoral and cellular immunity of hosts. Notably, they could significantly increase the expression of immune-associated cytokines, especially the IFN-γ. Due to the robust immunogenicity, thus DPs generated in infected mast cells could stimulate the robust protective immune reaction effectively to fight against lethal IAV re-challenge after vaccination, which result in the high survival, decreased lung injury as well as inhibition of viral replication and inflammatory response in lungs. This study is the first to illustrate and explore the safety, immunogenicity, and effectiveness of DPs arising in mast cells against influenza as favorable potential vaccination. The results provide insight into the advances of new prophylactic strategies to fight influenza by focusing on DPs generated in mast cells.

Cell-to-Cell Variation in Defective Virus Expression and Effects on Host Responses during Influenza Virus Infection.

Virus and host factors contribute to cell-to-cell variation in viral infections and determine the outcome of the overall infection. However, the extent of the variability at the single-cell level and how it impacts virus-host interactions at a system level are not well understood. To characterize the dynamics of viral transcription and host responses, we used single-cell RNA sequencing to quantify at multiple time points the host and viral transcriptomes of human A549 cells and primary bronchial epithelial cells infected with influenza A virus. We observed substantial variability in viral transcription between cells, including the accumulation of defective viral genomes (DVGs) that impact viral replication. We show (i) a correlation between DVGs and virus-induced variation of the host transcriptional program and (ii) an association between differential inductions of innate immune response genes and attenuated viral transcription in subpopulations of cells. These observations at the single-cell level improve our understanding of the complex virus-host interplay during influenza virus infection.IMPORTANCE Defective influenza virus particles generated during viral replication carry incomplete viral genomes and can interfere with the replication of competent viruses. These defective genomes are thought to modulate the disease severity and pathogenicity of an influenza virus infection. Different defective viral genomes also introduce another source of variation across a heterogeneous cell population. Evaluating the impact of defective virus genomes on host cell responses cannot be fully resolved at the population level, requiring single-cell transcriptional profiling. Here, we characterized virus and host transcriptomes in individual influenza virus-infected cells, including those of defective viruses that arise during influenza A virus infection. We established an association between defective virus transcription and host responses and validated interfering and immunostimulatory functions of identified dominant defective viral genome species in vitro This study demonstrates the intricate effects of defective viral genomes on host transcriptional responses and highlights the importance of capturing host-virus interactions at the single-cell level.

Vaccination of Macaques with DNA Followed by Adenoviral Vectors Encoding Simian Immunodeficiency Virus (SIV) Gag Alone Delays Infection by Repeated Mucosal Challenge with SIV.

Vaccines aimed at inducing T cell responses to protect against human immunodeficiency virus (HIV) infection have been under development for more than 15 years. Replication-defective adenovirus (rAd) vaccine vectors are at the forefront of this work and have been tested extensively in the simian immunodeficiency virus (SIV) challenge macaque model. Vaccination with rAd vectors coding for SIV Gag or other nonenvelope proteins induces T cell responses that control virus load but disappointingly is unsuccessful so far in preventing infection, and attention has turned to inducing antibodies to the envelope. However, here we report that Mauritian cynomolgus macaques (MCM), Macaca fascicularis, vaccinated with unmodified SIV gag alone in a DNA prime followed by an rAd boost exhibit increased protection from infection by repeated intrarectal challenge with low-dose SIVmac251. There was no evidence of infection followed by eradication. A significant correlation was observed between cytokine expression by CD4 T cells and delayed infection. Vaccination with gag fused to the ubiquitin gene or fragmented, designed to increase CD8 magnitude and breadth, did not confer resistance to challenge or enhance immunity. On infection, a significant reduction in peak virus load was observed in all vaccinated animals, including those vaccinated with modified gag These findings suggest that a nonpersistent viral vector vaccine coding for internal virus proteins may be able to protect against HIV type 1 (HIV-1) infection. The mechanisms are probably distinct from those of antibody-mediated virus neutralization or cytotoxic CD8 cell killing of virus-infected cells and may be mediated in part by CD4 T cells.IMPORTANCE The simian immunodeficiency virus (SIV) macaque model represents the best animal model for testing new human immunodeficiency virus type 1 (HIV-1) vaccines. Previous studies employing replication-defective adenovirus (rAd) vectors that transiently express SIV internal proteins induced T cell responses that controlled virus load but did not protect against virus challenge. However, we show for the first time that SIV gag delivered in a DNA prime followed by a boost with an rAd vector confers resistance to SIV intrarectal challenge. Other partially successful SIV/HIV-1 protective vaccines induce antibody to the envelope and neutralize the virus or mediate antibody-dependent cytotoxicity. Induction of CD8 T cells which do not prevent initial infection but eradicate infected cells before infection becomes established has also shown some success. In contrast, the vaccine described here mediates resistance by a different mechanism from that described above, which may reflect CD4 T cell activity. This could indicate an alternative approach for HIV-1 vaccine development.

Flu DRiPs in MHC Class I Immunosurveillance.

Since the publication of the DRiP (defective ribosomal product) hypothesis in 1996, numerous studies have addressed the contribution of DRiPs to generating viral antigenic peptides for CD8+ T cell immunosurveillance. Here, we review studies characterizing the generation of antigenic peptides from influenza A virus encoded DRiPs, discuss the many remaining mysteries regarding the nature of their co-translational generation, and speculate on where the future might lead.

Comparison of vaccination with rhesus CMV (RhCMV) soluble gB with a RhCMV replication-defective virus deleted for MHC class I immune evasion genes in a RhCMV challenge model.

A human cytomegalovirus (HCMV) vaccine to prevent infection and/or reduce disease associated with congenital infection or visceral disease in transplant recipients is a high priority, but has remained elusive. We created a disabled infectious single cycle rhesus CMV (RhCMV) deleted for glycoprotein L (gL) and the MHC class I immune evasion genes Rh178 and Rh182-189, and restored its epithelial cell tropism by inserting the Rh128-131A genes. The resulting virus, RhCMVRΔgL/178/182-189, was used to vaccinate rhesus monkeys intramuscularly and was compared with vaccination of animals with soluble RhCMV glycoprotein B (gB) in alum/monophosphoryl lipid A or with PBS as a control. At 4 weeks after the second vaccination, an increased frequency of RhCMV-specific CD8 T cells was detected in animals vaccinated with the RhCMVRΔgL/178/182-189 vaccine compared to animals vaccinated with soluble gB. In contrast, monkeys vaccinated with soluble gB had 20-fold higher gB antibody titers than animals vaccinated with RhCMVRΔgL/178/182-189. Titers of neutralizing antibody to RhCMV infection of fibroblasts were higher in animals vaccinated with gB compared with RhCMVRΔgL/178/182-189. Following vaccination, monkeys were challenged subcutaneously with RhCMV UCD59, a low passage virus propagated in monkey kidney epithelial cells. All animals became infected after challenge; however, the frequency of RhCMV detection in the blood was reduced in monkeys vaccinated with soluble gB compared with those vaccinated with RhCMVRΔgL/178/182-189. The frequency of challenge virus shedding in the urine and saliva and the RhCMV copy number shed at these sites was not different in animals vaccinated with RhCMVRΔgL/178/182-189 or soluble gB compared with those that received PBS before challenge. Although the RhCMVRΔgL/178/182-189 vaccine was superior in inducing cellular immunity to RhCMV, it induced lower titers of neutralizing antibody and antibody to gB than the soluble gB vaccine; after challenge, animals vaccinated with soluble gB had a lower frequency of virus detection in the blood than those vaccinated with RhCMVRΔgL/178/182-189.

A Single Amino Acid Substitution within the Paramyxovirus Sendai Virus Nucleoprotein Is a Critical Determinant for Production of Interferon-Beta-Inducing Copyback-Type Defective Interfering Genomes.

One of the first defenses against infecting pathogens is the innate immune system activated by cellular recognition of pathogen-associated molecular patterns (PAMPs). Although virus-derived RNA species, especially copyback (cb)-type defective interfering (DI) genomes, have been shown to serve as real PAMPs, which strongly induce interferon-beta (IFN-ß) during mononegavirus infection, the mechanisms underlying DI generation remain unclear. Here, for the first time, we identified a single amino acid substitution causing production of cbDI genomes by successful isolation of two distinct types of viral clones with cbDI-producing and cbDI-nonproducing phenotypes from the stock Sendai virus (SeV) strain Cantell, which has been widely used in a number of studies on antiviral innate immunity as a representative IFN-ß-inducing virus. IFN-ß induction was totally dependent on the presence of a significant amount of cbDI genome-containing viral particles (DI particles) in the viral stock, but not on deficiency of the IFN-antagonistic viral accessory proteins C and V. Comparison of the isolates indicated that a single amino acid substitution found within the N protein of the cbDI-producing clone was enough to cause the emergence of DI genomes. The mutated N protein of the cbDI-producing clone resulted in a lower density of nucleocapsids than that of the DI-nonproducing clone, probably causing both production of the DI genomes and their formation of a stem-loop structure, which serves as an ideal ligand for RIG-I. These results suggested that the integrity of mononegaviral nucleocapsids might be a critical factor in avoiding the undesirable recognition of infection by host cells.IMPORTANCE The type I interferon (IFN) system is a pivotal defense against infecting RNA viruses that is activated by sensing viral RNA species. RIG-I is a major sensor for infection with most mononegaviruses, and copyback (cb)-type defective interfering (DI) genomes have been shown to serve as strong RIG-I ligands in real infections. However, the mechanism underlying production of cbDI genomes remains unclear, although DI genomes emerge as the result of an error during viral replication with high doses of viruses. Sendai virus has been extensively studied and is unique in that its interaction with innate immunity reveals opposing characteristics, such as high-level IFN-ß induction and strong inhibition of type I IFN pathways. Our findings provide novel insights into the mechanism of production of mononegaviral cbDI genomes, as well as virus-host interactions during innate immunity.

PACT- and RIG-I-Dependent Activation of Type I Interferon Production by a Defective Interfering RNA Derived from Measles Virus Vaccine.

UNLABELLED: The live attenuated measles virus vaccine is highly immunostimulatory. Identification and characterization of its components that activate the innate immune response might provide new strategies and agents for the rational design and development of chemically defined adjuvants. In this study, we report on the activation of type I interferon (IFN) production by a defective interfering (DI) RNA isolated from the Hu-191 vaccine strain of measles virus. We found that the Hu-191 virus induced IFN-ß much more potently than the Edmonston strain. In the search for IFN-inducing species in Hu-191, we identified a DI RNA specifically expressed by this strain. This DI RNA, which was of the copy-back type, was predicted to fold into a hairpin structure with a long double-stranded stem region of 206 bp, and it potently induced the expression of IFN-ß. Its IFN-ß-inducing activity was further enhanced when both cytoplasmic RNA sensor RIG-I and its partner, PACT, were overexpressed. On the contrary, this activity was abrogated in cells deficient in PACT or RIG-I. The DI RNA was found to be associated with PACT in infected cells. In addition, both the 5'-di/triphosphate end and the double-stranded stem region on the DI RNA were essential for its activation of PACT and RIG-I. Taken together, our findings support a model in which a viral DI RNA is sensed by PACT and RIG-I to initiate an innate antiviral response. Our work might also provide a foundation for identifying physiological PACT ligands and developing novel adjuvants or antivirals. IMPORTANCE: The live attenuated measles virus vaccine is one of the most successful human vaccines and has largely contained the devastating impact of a highly contagious virus. Identifying the components in this vaccine that stimulate the host immune response and understanding their mechanism of action might help to design and develop better adjuvants, vaccines, antivirals, and immunotherapeutic agents. We identified and characterized a defective interfering RNA from the Hu-191 vaccine strain of measles virus which has safely been used in millions of people for many years. We further demonstrated that this RNA potently induces an antiviral immune response through cellular sensors of viral RNA known as PACT and RIG-I. Similar types of viral RNA that bind with and activate PACT and RIG-I might retain the immunostimulatory property of measles virus vaccines but would not induce adaptive immunity. They are potentially useful as chemically defined vaccine adjuvants, antivirals, and immunostimulatory agents.

An HPV-E6/E7 immunotherapy plus PD-1 checkpoint inhibition results in tumor regression and reduction in PD-L1 expression.

We have investigated if immunotherapy against human papilloma virus (HPV) using a viral gene delivery platform to immunize against HPV 16 genes E6 and E7 (Ad5 [E1-, E2b-]-E6/E7) combined with programmed death-ligand 1 (PD-1) blockade could increase therapeutic effect as compared to the vaccine alone. Ad5 [E1-, E2b-]-E6/E7 as a single agent induced HPV-E6/E7 cell-mediated immunity. Immunotherapy using Ad5 [E1-, E2b-]-E6/E7 resulted in clearance of small tumors and an overall survival benefit in mice with larger established tumors. When immunotherapy was combined with immune checkpoint blockade, an increased level of anti-tumor activity against large tumors was observed. Analysis of the tumor microenvironment in Ad5 [E1-, E2b-]-E6/E7 treated mice revealed elevated CD8(+) tumor infiltrating lymphocytes (TILs); however, we observed induction of suppressive mechanisms such as programmed death-ligand 1 (PD-L1) expression on tumor cells and an increase in PD-1(+) TILs. When Ad5 [E1-, E2b-]-E6/E7 immunotherapy was combined with anti-PD-1 antibody, we observed CD8(+) TILs at the same level but a reduction in tumor PD-L1 expression on tumor cells and reduced PD-1(+) TILs providing a mechanism by which combination therapy favors a tumor clearance state and a rationale for pairing antigen-specific vaccines with checkpoint inhibitors in future clinical trials.

Accumulation of defective interfering viral particles in only a few passages in Vero cells attenuates mumps virus neurovirulence.

Immunization programs have implemented live attenuated mumps vaccines which reduced mumps incidence ≥97%. Some of the vaccine strains were abandoned due to unwanted side effects and the genetic marker of attenuation has not been identified so far. Our hypothesis was that non-infectious viral particles, in particular defective interfering particles (DIPs), contribute to neuroattenuation. We showed that non-infectious particles of the mumps vaccine L-Zagreb attenuated neurovirulence of wild type mumps virus 9218/Zg98. Then, we attenuated recent wild type mumps virus MuVi/Zagreb.HRV/28.12 in Vero cells through 16 passages but already the fifth passage (p5) showed accumulation of DIPs and attenuated neurovirulence in a newborn rat model when compared to the second passage (p2). Sequence analysis of the p2 and p5 revealed a single mutation in the 5' untranslated region of the HN gene. Analysis of the expression level of the HN protein showed that this mutation does not affect the expression of the protein. We conclude that the passages of MuVi/Zagreb.HRV/28.12 in Vero cells for only three passages accumulated DIPs which attenuate neurovirulence. These findings reveal DIPs as a very promising and general neuroattenuating factor which should be considered in the rational design of the new mumps vaccine.

Chimpanzee adenovirus vaccine generates acute and durable protective immunity against ebolavirus challenge.

Ebolavirus disease causes high mortality, and the current outbreak has spread unabated through West Africa. Human adenovirus type 5 vectors (rAd5) encoding ebolavirus glycoprotein (GP) generate protective immunity against acute lethal Zaire ebolavirus (EBOV) challenge in macaques, but fail to protect animals immune to Ad5, suggesting natural Ad5 exposure may limit vaccine efficacy in humans. Here we show that a chimpanzee-derived replication-defective adenovirus (ChAd) vaccine also rapidly induced uniform protection against acute lethal EBOV challenge in macaques. Because protection waned over several months, we boosted ChAd3 with modified vaccinia Ankara (MVA) and generated, for the first time, durable protection against lethal EBOV challenge.

[Anti-tumor effect of Sendai virus Tianjin strain defective interfering particles on tumor-bearing mice].

OBJECTIVE: To explore the anti-tumor effect and its mechanism of Sendai virus Tianjin strain defective interfering particles (DIP) on mouse models of colon carcinoma. METHODS: CT26 cells (5×10(6)/0.1 ml) were subcutaneously injected into the back of Bal B/c mice to establish murine colon carcinoma model. After the tumors reached 5 mm in diameter, the mice were randomly divided into Tianjin strain DIP group and saline control group. The former was intratumorally injected with Tianjin strain DIP (0.1 ml) once a day on day 4, 7, 10 and 13 after CT26 cell inoculation. The latter was intratumorally injected with the same volume of saline. Tumor volume and survival rate of the mice were calculated to confirm the anti-tumor effect of DIP. Flow cytometry and ELISA were used to examine the maturation and release of cytokines IL-6, IFN-α and TNF-α from murine myeloid dendritic cells (DCs) induced by Tianjin strain DIP. Moreover, real-time RT-PCR and immunohistochemistry were performed to identify whether the Tianjin strain DIP could induce infiltration of CD11c(+) DCs, CD4(+) and CD8(+) T cells in the tumors. RESULTS: On day 22 after CT26 cell inoculation, the average tumor volume of the Tianjin strain DIP group was (33.2 ± 2.0) mm(3), significantly smaller than that of the control group [(2 376.0 ± 130.8)mm(3), P < 0.01]. On day 50 after CT26 cell inoculation, the survival rate of mice was 90.0% in the Tianjin strain DIP group, much higher than that of the control group (30.0%, P < 0.01). Flow cytometry analysis showed that the expression of markers of DCs maturation, including CD40, CD80 and CD86, was dose-dependently increased by DIP or intact virus. No statistically significant difference was found betweent the DIP and intact virus groups. ELISA results showed that DIP could stimulate the secretion of IL-6, IFN-α and TNF-α from mouse DCs. The secretion of all of the cytokines was dose-dependently increased by DIP or intact virus. Real-time RT-PCR revealed that the expression of CD4, CD8 and CD11c mRNAs was increased in tumors treated with DIP compared with that of the saline group at all time points. Moreover, the expression level of all of them remained maximal at 120 h after the last treatment. Immunohistochemical staining revealed that the ratios of CD4(+), CD8(+) T cells or CD11c(+) DCs to total cells were (21.60 ± 1.49)%, (22.12 ± 2.84)% and (23.05 ± 2.91)%, respectively, in the DIP-treated tumors. In the tumors treated by saline, the ratios were (2.62 ± 0.60)%, (4.05 ± 0.12)% and (3.10 ± 0.09)%, respectively. The difference between experimental group and control group had statistical significance. CONCLUSIONS: Tianjin strain DIP may exert anti-tumor effect on tumor-bearing mice. The mechanism is related with the antitumor immunity induced by DCs and T cells.

Adenovirus E1B 19-kilodalton protein modulates innate immunity through apoptotic mimicry.

UNLABELLED: Cells that undergo apoptosis in response to chemical or physical stimuli repress inflammatory reactions, but cells that undergo nonapoptotic death in response to such stimuli lack this activity. Whether cells dying from viral infection exhibit a cell death-type modulatory effect on inflammatory reactions is unknown. We compared the effects on macrophage inflammatory responses of cells dying an apoptotic or a nonapoptotic death as a result of adenoviral infection. The results were exactly opposite to the predictions from the conventional paradigm. Cells dying by apoptosis induced by infection with an adenovirus type 5 (Ad5) E1B 19-kilodalton (E1B 19K) gene deletion mutant did not repress macrophage NF-κB activation or cytokine responses to proinflammatory stimuli, whereas cells dying a nonapoptotic death from infection with E1B 19K-competent, wild-type Ad5 repressed these macrophage inflammatory responses as well as cells undergoing classical apoptosis in response to chemical injury. The immunorepressive, E1B 19K-related cell death activity depended upon direct contact of the virally infected corpses with responder macrophages. Replacement of the viral E1B 19K gene with the mammalian Bcl-2 gene in cis restored the nonapoptotic, immunorepressive cell death activity of virally infected cells. These results define a novel function of the antiapoptotic, adenoviral E1B 19K protein that may limit local host innate immune inflammation during accumulation of virally infected cells at sites of infection and suggest that E1B 19K-deleted, replicating adenoviral vectors might induce greater inflammatory responses to virally infected cells than E1B 19K-positive vectors, because of the net effect of their loss-of-function mutation. IMPORTANCE: We observed that cells dying a nonapoptotic cell death induced by adenovirus infection repressed macrophage proinflammatory responses while cells dying by apoptosis induced by infection with an E1B 19K deletion mutant virus did not repress macrophage proinflammatory responses and enhanced some cytokine responses. Our results define a new function of the antiapoptotic, adenoviral protein E1B 19K, which we have termed "apoptotic mimicry." Our studies suggest the possibility that the presence or absence of this E1B 19K function could alter the immunological outcome of both natural and therapeutic adenoviral infections. For example, emerging, highly immunopathogenic adenovirus serotypes might induce increased host inflammatory responses as a result of altered E1B 19K function or expression. It is also possible that engineered variations in E1B 19K expression/function could be created during adenovirus vector design that would increase the therapeutic efficacy of replicating adenovirus vectors for vaccines or oncolytic viral targeting of neoplastic cells.

Highly immunostimulatory RNA derived from a Sendai virus defective viral genome.

Defective viral genomes (DVGs) are generated during virus replication. DVGs bearing complementary ends are strong inducers of dendritic cell (DC) maturation and of the expression of antiviral and pro-inflammatory cytokines by triggering signaling of the RIG-I family of intracellular pattern recognition receptors. Our data show that DCs stimulated with virus containing DVGs have an enhanced ability to activate human T cells and can induce adaptive immunity in mice. In addition, we describe the generation of a short Sendai virus (SeV)-derived DVG RNA (DVG-324) that maintains strong immunostimulatory activity in vitro and in vivo. DVG-324 induced high levels of Ifnb expression when transfected into cells and triggered fast expression of pro-inflammatory cytokines and mobilization of dendritic cells when injected into the footpad of mice. Importantly, DVG-324 enhanced the production of antibodies to a prototypic vaccine after a single intramuscular immunization in mice. Notably, the pro-inflammatory cytokine profile induced by DVG-324 was different from that induced by poly I:C, the only viral RNA analog currently used as an immunostimulant in vivo, suggesting a distinct mechanism of action. SeV-derived oligonucleotides represent novel alternatives to be harnessed as potent adjuvants for vaccination.

The novel replication-defective vaccinia virus (Tiantan strain)-based hepatitis C virus vaccine induces robust immunity in macaques.

The induction of a robust neutralizing antibody (nAb) response is likely to be as essential as specific cell-mediated immunity (CMI) against multiple antigens for the development of effective preventive and therapeutic vaccines against hepatitis C virus (HCV) infection in humans. To date, no data on the immunogenicity of the replication-defective vaccinia virus (derived from the Tiantan strain) (rNTV)-based HCV vaccine in primates have been reported. This study describes in detail the immunogenicity of various vaccine candidates in rhesus macaques, including rNTV-based and replication-defective recombinant adenoviral (rAd)-based HCV vaccines, as well as HCV pseudotyped virus-like particles (HCVpp). Our data showed that rAd-HCV vaccine boosting induced robust CMI, while priming or boosting with HCVpp enhanced the antigen-specific nAb response after rAd-HCV vaccination; however, CMI was not enhanced. Vaccination includes rNTV-HCV priming induced robust antigen-specific antibody, particularly nAbs, and CMI responses. Furthermore, more robust and longer-lasting CMI and higher cytokine levels (both Th1 and Th2 types, especially IFN-γ) resulted from boosting with rAd-HCV. We conclude that the rNTV-based HCV vaccine induces robust nAbs and CMI when combined with a heterogeneous primer-booster strategy, which shows promise for development of a human HCV vaccine.

An Epstein-Barr virus mutant produces immunogenic defective particles devoid of viral DNA.

Virus-like particles (VLPs) from hepatitis B and human papillomaviruses have been successfully used as preventative vaccines against these infectious agents. These VLPs consist of a self-associating capsid polymer formed from a single structure protein and are devoid of viral DNA. Since virions from herpesviruses consist of a large number of molecules of viral and cellular origin, generating VLPs from a subset of these would be a particularly arduous task. Therefore, we have adopted an alternative strategy that consists of producing DNA-free defective virus particles in a cell line infected by a herpesvirus mutant incapable of packaging DNA. We previously reported that an Epstein-Barr virus (EBV) mutant devoid of the terminal repeats (ΔTR) that act as packaging signals in herpesviruses produces substantial amounts of VLPs and of light particles (LPs). However, ΔTR virions retained some infectious genomes, and although these mutants had lost their transforming abilities, this poses potential concerns for clinical applications. Therefore, we have constructed a series of mutants that lack proteins involved in maturation and assessed their ability to produce viral DNA-free VLP/LPs. Some of the introduced mutations were deleterious for capsid maturation and virus production. However, deletion of BFLF1/BFRF1A or of BBRF1 resulted in the production of DNA-free VLPs/LPs. The ΔBFLF1/BFRF1A viruses elicited a potent CD4(+) T-cell response that was indistinguishable from the one obtained with wild-type controls. In summary, the defective particles produced by the ΔBFLF1/BFRF1A mutant fulfill the criteria of efficacy and safety expected from a preventative vaccine.

Immunization with apoptotic pseudovirus transduced cells induces both cellular and humoral responses: a proof of concept study in macaques.

Dendritic cells are able to present viral antigens to T-cells after uptake of apoptotic bodies derived from virus-infected cells. Immunization with virus-infected apoptotic cells was previously shown to induce HIV-specific immune responses in mice. Here we evaluate the safety and immunogenicity of immunization with activated apoptotic cells in non-human primates using autologous T-cells infected with replication defective VSV pseudotyped SIV(mac239)Δenv. Animals were immunized with γ-irradiated activated T-cells carrying the VSVenvSIV(mac239)Δenv pseudovirus. SIV Gag-specific cellular immune responses were induced as early as two weeks after the first immunization eliciting a biased IFN-γ and IL-2 response. In addition, induction of SIV Gag-specific antibody responses and high titer neutralizing activity against the SIV pseudovirus harboring a VSV-env were detected after two immunizations. The vaccinated group and a control group of Chinese rhesus macaques were intravenously challenged with pathogenic SIV(mac251.) All animals became infected, but SIV-replication was effectively suppressed (below 100 copies/ml) in several animals in both groups. However the group immunized with apoptotic cells revealed better preservation of the gut CD4(+) T-cell compartment. Viral control was inversely correlated with an early (4 weeks) but transient increase in the percentage of Ki67(+)CD4(+) peripheral blood T-cells (Spearman -0.73). We here show that immunizations with activated apoptotic lymphocytes expressing transduced SIV genes result in induction of both cellular and humoral immune responses. This study provides evidence for an immunological principle demonstrating that certain apoptotic cells can be considered as carriers of antigens directing immune responses in macaques.

Differential recognition of viral RNA by RIG-I.

Retinoic acid inducible gene I (RIG-I) is a pattern recognition receptor (PRR) responsible for detection of nucleic acids from pathogens in the cytoplasm of infected cells and induction of type I interferon (IFN). RIG-I -specific pathogen associated molecular patterns (PAMPs) are characterized by RNA molecules with a 5'-triphosphate (5'-ppp) group and partial double-stranded composition. Although many RNA molecules capable of activating RIG-I have been described, the exact nature of viral RNAs which are responsible for triggering RIG-I activity during the course of an infection has not been extensively explored and the specificity of RIG-I for various viral RNA molecules remains largely unknown. By examining endogenous RIG-I/RNA complexes in influenza virus and Sendai virus infected cells we were able to identify viral RNA molecules which specifically associated with RIG-I during infection. We showed that in Sendai virus infected cells, RIG-I specifically and preferentially associated with the copy-back defective interfering (DI) particle RNA and not with the full-length Sendai virus genome or Sendai virus encoded mRNAs. In influenza virus infected cells RIG-I also preferentially associated with DI RNAs as well as with the shorter genomic segments.

Cocaine analog coupled to disrupted adenovirus: a vaccine strategy to evoke high-titer immunity against addictive drugs.

Based on the concept that anticocaine antibodies could prevent inhaled cocaine from reaching its target receptors in the brain, an effective anticocaine vaccine could help reverse cocaine addiction. Leveraging the knowledge that E1(-)E3(-) adenovirus (Ad) gene transfer vectors are potent immunogens, we have developed a novel vaccine platform for addictive drugs by covalently linking a cocaine analog to the capsid proteins of noninfectious, disrupted Ad vector. The Ad-based anticocaine vaccine evokes high-titer anticocaine antibodies in mice sufficient to completely reverse, on a persistent basis, the hyperlocomotor activity induced by intravenous administration of cocaine.

Complementing defective viruses that express separate paramyxovirus glycoproteins provide a new vaccine vector approach.

Replication-defective vaccine vectors based on vesicular stomatitis virus (VSV) lacking its envelope glycoprotein gene (G) are highly effective in animal models. However, such ΔG vectors are difficult to grow because they require complementation with the VSV G protein. In addition, the complementing G protein induces neutralizing antibodies in animals and thus limits multiple vector applications. In the process of generating an experimental Nipah virus (a paramyxovirus) vaccine, we generated two defective VSVΔG vectors, each expressing one of the two Nipah virus (NiV) glycoproteins (G and F) that are both required for virus entry to host cells. These replication-defective VSV vectors were effective at generating NiV neutralizing antibody in mice. Most interestingly, we found that these two defective viruses could be grown together and passaged in tissue culture cells in the absence of VSV G complementation. This mixture of complementing defective viruses was also highly effective at generating NiV neutralizing antibody in animals. This novel approach to growing and producing a vaccine from two defective viruses could be generally applicable to vaccine production for other paramyxoviruses or for other viruses where the expression of at least two different proteins is required for viral entry. Such an approach minimizes biosafety concerns that could apply to single, replication-competent VSV recombinants expressing all proteins required for infection.