Synthetic double-stranded RNA poly(I:C) as a potent peptide vaccine adjuvant: therapeutic activity against human cervical cancer in a rodent model

Due to the inherent lack of immunogenicity of peptides, it is generally recognized that the strong inflammatory signals that are required to elicit specific responses against peptide-based therapeutic tumor vaccines may not be provided by the standard/conventional vaccine adjuvants. In this study, we have demonstrated dsRNA in the form of synthetic pI:C as a potent adjuvant to enhance the specific anti-tumor immune responses against a peptide-based vaccine. When complexed with an MHC I-restricted minimal peptide epitope derived from the HPV 16 E7 protein, the resulting pI:C/E7_49–57 molecular complex induced strong E7_49–57-specific CTL responses that caused significant regressions of model human cervical cancer tumors pre-established in mice. In addition, although the proportion of DCs in tumor-bearing mice was significantly decreased when compared to that in naïve mice, immunization with pI:C/E7_49–57 restored the proportion of DCs in tumor-bearing mice. Double-stranded RNA may hold a great potential as an adjuvant to induce cellular immune responses for tumor immunotherapy.     

Seasonal trivalent inactivated influenza vaccine protects against 1918 Spanish influenza virus infection in ferrets

The influenza virus H1N1 pandemic of 1918 was one of the worst medical catastrophes in human history. Recent studies have demonstrated that the hemagglutinin (HA) protein of the 1918 virus and 2009 H1N1 pandemic virus [A(H1N1)pdm09], the latter now a component of the seasonal trivalent inactivated influenza vaccine (TIV), share cross-reactive antigenic determinants. In this study, we demonstrate that immunization with the 2010-2011 seasonal TIV induces neutralizing antibodies that cross-react with the reconstructed 1918 pandemic virus in ferrets. TIV-immunized ferrets subsequently challenged with the 1918 virus displayed significant reductions in fever, weight loss, and virus shedding compared to these parameters in nonimmune control ferrets. Seasonal TIV was also effective in protecting against the lung infection and severe lung pathology associated with 1918 virus infection. Our data demonstrate that prior immunization with contemporary TIV provides cross-protection against the 1918 virus in ferrets. These findings suggest that exposure to A(H1N1)pdm09 through immunization may provide protection against the reconstructed 1918 virus which, as a select agent, is considered to pose both biosafety and biosecurity threats.     

Yellow fever vaccine protects mice against Zika virus infection

Zika virus (ZIKV) emerged as an important infectious disease agent in Brazil in 2016. Infection usually leads to mild symptoms, but severe congenital neurological disorders and Guillain-Barré syndrome have been reported following ZIKV exposure. Creating an effective vaccine against ZIKV is a public health priority. We describe the protective effect of an already licensed attenuated yellow fever vaccine (YFV, 17DD) in type-I interferon receptor knockout mice (A129) and immunocompetent BALB/c and SV-129 (A129 background) mice infected with ZIKV. YFV vaccination provided protection against ZIKV, with decreased mortality in A129 mice, a reduction in the cerebral viral load in all mice, and weight loss prevention in BALB/c mice. The A129 mice that were challenged two and three weeks after the first dose of the vaccine were fully protected, whereas partial protection was observed five weeks after vaccination. In all cases, the YFV vaccine provoked a substantial decrease in the cerebral viral load. YFV immunization also prevented hippocampal synapse loss and microgliosis in ZIKV-infected mice. Our vaccine model is T cell-dependent, with AG129 mice being unable to tolerate immunization (vaccination is lethal in this mouse model), indicating the importance of IFN-γ in immunogenicity. To confirm the role of T cells, we immunized nude mice that we demonstrated to be very susceptible to infection. Immunization with YFV and challenge 7 days after booster did not protect nude mice in terms of weight loss and showed partial protection in the survival curve. When we evaluated the humoral response, the vaccine elicited significant antibody titers against ZIKV; however, it showed no neutralizing activity in vitro and in vivo. The data indicate that a cell-mediated response promotes protection against cerebral infection, which is crucial to vaccine protection, and it appears to not necessarily require a humoral response. This protective effect can also be attributed to innate factors, but more studies are needed to strengthen this hypothesis. Our findings open the way to using an available and inexpensive vaccine for large-scale immunization in the event of a ZIKV outbreak.     

Quantification of poly(I:C)-mediated protection against genital herpes simplex virus type 2 infection

Alternative strategies for controlling the growing herpes simplex virus type 2 (HSV-2) epidemic are needed. A novel class of immunomodulatory microbicides has shown promise as antiherpetics, including intravaginally applied CpG-containing oligodeoxynucleotides that stimulate toll-like receptor 9 (TLR9). In the current study, we quantified protection against experimental genital HSV-2 infection provided by an alternative nucleic acid-based TLR agonist, polyinosine-poly(C) (PIC) (TLR3 agonist). Using a protection quantification paradigm, groups of mice were PIC treated and then subdivided into groups challenged with escalating doses of HSV-2. Using this paradigm, a temporal window of PIC efficacy for single applications was defined as 1 day prior to (prophylactic) through 4 h after (therapeutic) viral challenge. PIC treatment within this window protected against 10-fold-higher HSV-2 challenges, as indicated by increased 50% infectious dose values relative to those for vehicle-treated controls. Disease resolution and survival were significantly enhanced by repetitive PIC doses. Using optimal PIC regimens, cytokine induction was evaluated in murine vaginal lavages and in human vaginal epithelial cells. Similar induction patterns were observed, with kinetics that explained the limited durability of PIC-afforded protection. Daily PIC delivery courses did not generate sustained cytokine levels in murine vaginal fluids that would be indicative of local immunotoxicity. No evidence of immunotoxicity was observed in selected organs that were analyzed following repetitive vaginal PIC doses. Animal and in vitro data indicate that PIC may prove to be a valuable preventative microbicide and/or therapeutic agent against genital herpes by increasing resistance to HSV-2 and enhancing disease resolution following a failure of prevention.     

Protection of inactivated influenza virus vaccine against lethal influenza virus infection in diabetic mice

Influenza virus infection frequently causes complications and some excess mortality in the patients with diabetes. Vaccination is an effective measure to prevent influenza virus infection. In this paper, antibody response and protection against influenza virus infection induced by vaccination were studied in mouse model of diabetes. Healthy and diabetic BALB/c mice were immunized once or twice with inactivated influenza virus vaccine at various dosages. Four weeks after the first immunization or 1 week after the second immunization, the mice were challenged with influenza virus at a lethal dose. The result showed that the antibody responses in diabetic mice were inhibited. Immunization once with high dose or twice with low dose of vaccine provided full protection against lethal influenza virus challenge in diabetic mice, however, in healthy mice, immunization only once with low dose provided a full protection.     

Proteins induced by the double-stranded interferon inducer poly(I).poly(C)

The possible pathways for realization of antiviral activity of interferon inducer poly (I).poly(C) have been studied. The stimulating effect of interferon inducer on the net protein synthesis in human M19 fibroblasts has been demonstrated. Compositions of the specific proteins induced by poly(I).poly(C) or interferon in human M19 fibroblasts and in monkey cells 4647 have been analyzed by electrophoresis technique. The data obtained suggest the existence of common gene products for interferon and ds-inducer. The ds-inducer requires the synthesis of lesser amounts of proteins for realization of its biological activity as compared with interferon.     

Dissociation of double-stranded poly(I) . poly(C) by cis-diammine-dichloro-Pt(II).

The covalent binding of cis-Pt(NH3)2Cl2 on the double stranded poly(I) . poly(C) induced an irreversible dissociation of the two strands. This dissociation was evidenced mainly by poly(I)-Agarose affinity chromatography which allowed to recover free strands of cis-Pt(NH3)2Cl2-poly(I) from a cis-Pt(NH3)2Cl2-poly(I) . poly(C) complex, by density equilibrium centrifugation where free poly(C) could be isolated, and by acid titrations of the metal-poly(I) . poly(C) complexes. The separation of the two strands of the polyribonucleotide upon cis-Pt(NH3)2Cl2 fixation was shown not to exceed 90--95%. A dissociation curve of the polynucleotide double helix as a function of the amount of bound cis-Pt(NH3)2Cl2 was determined and was shown to be of a characteristic cooperative effect. The fixation of the paltinum compound to poly(I) . poly(C) seemed also to be cooperative.     

An antiviral state induced in Chinook salmon embryo cells (CHSE-214) by transfection with the double-stranded RNA poly I:C

Type I interferons (IFN alpha and beta) convert vertebrate cells into an antiviral state by inducing expression of proteins that inhibit virus replication. In humans and mice, Mx proteins constitute one family of interferon-induced antiviral proteins. Mx genes have recently been cloned from Atlantic salmon and rainbow trout. Moreover, double-stranded RNA (dsRNA) and type I IFN-like activity have been shown to induce Mx protein in salmonid cells. Chinook salmon embryo cells (CHSE-214 cells) have been suggested to have a defect in the IFN-system because the dsRNA polyinosinic polycytidylic acid (poly I:C) failed to induce an antiviral state in the cells. We have studied this phenomenon more closely in the present work. CHSE-214 cells were either transfected with poly I:C or incubated with poly I:C without transfection reagent. The cells were then studied for Mx protein expression and protection against infectious pancreatic necrosis virus (IPNV) infection. The results showed that cells transfected with poly I:C were protected from IPNV infection, whilst cells incubated with poly I:C were not protected. Cells transfected with the double-stranded DNA poly dI:dC were also not protected against IPNV. Mx protein was expressed in CHSE-214 cells upon transfection with poly I:C, but not after incubation with poly I:C alone. Stimulation of CHSE-214 cells with supernatants from cells transfected with poly I:C, induced protection against IPNV, indicating production of type I IFN-like activity. These results suggest that CHSE-214 cells in fact are able to produce type I IFN, but may have defects in the mechanisms mediating uptake of poly I:C or may degrade unprotected poly I:C.     

Muramyl dipeptide induces NOD2-dependent Ly6C(high) monocyte recruitment to the lungs and protects against influenza virus infection

Bacterial peptidoglycan-derived muramyl dipeptide (MDP) and derivatives have long-recognized antiviral properties but their mechanism of action remains unclear. In recent years, the pattern-recognition receptor NOD2 has been shown to mediate innate responses to MDP. Here, we show that MDP treatment of mice infected with Influenza A virus (IAV) significantly reduces mortality, viral load and pulmonary inflammation in a NOD2-dependent manner. Importantly, the induction of type I interferon (IFN) and CCL2 chemokine was markedly increased in the lungs following MDP treatment and correlated with a NOD2-dependent enhancement in circulating monocytes. Mechanistically, the protective effect of MDP could be explained by the NOD2-dependent transient increase in recruitment of Ly6C(high) "inflammatory" monocytes and, to a lesser extent, neutrophils to the lungs. Indeed, impairment in both Ly6C(high) monocyte recruitment and survival observed in infected Nod2-/- mice treated with MDP was recapitulated in mice deficient for the chemokine receptor CCR2 required for CCL2-mediated Ly6C(high) monocyte migration from the bone marrow into the lungs. MDP-induced pulmonary monocyte recruitment occurred normally in IAV-infected and MDP-treated Ips-1-/- mice. However, IPS-1 was required for improved survival upon MDP treatment. Finally, mycobacterial N-glycolyl MDP was more potent than N-acetyl MDP expressed by most bacteria at reducing viral burden while both forms of MDP restored pulmonary function following IAV challenge. Overall, our work sheds light on the antiviral mechanism of a clinically relevant bacterial-derived compound and identifies the NOD2 pathway as a potential therapeutic target against IAV.     

TLR3 is essential for the induction of protective immunity against Punta Toro Virus infection by the double-stranded RNA (dsRNA), poly(I:C12U), but not Poly(I:C): differential recognition of synthetic dsRNA molecules

In the wake of RNA virus infections, dsRNA intermediates are often generated. These viral pathogen-associated molecular patterns can be sensed by a growing number of host cell cytosolic proteins and TLR3, which contribute to the induction of antiviral defenses. Recent evidence indicates that melanoma differentiation-associated gene-5 is the prominent host component mediating IFN production after exposure to the dsRNA analog, poly(I:C). We have previously reported that Punta Toro virus (PTV) infection in mice is exquisitely sensitive to treatment with poly(I:C(12)U), a dsRNA analog that has a superior safety profile while maintaining the beneficial activity of the parental poly(I:C) in the induction of innate immune responses. The precise host factor(s) mediating protective immunity following its administration remain to be elucidated. To assess the role of TLR3 in this process, mice lacking the receptor were used to investigate the induction of protective immunity, type I IFNs, and IL-6 following treatment. Unlike wild-type mice, those lacking TLR3 were not protected against PTV infection following poly(I:C(12)U) therapy and failed to produce IFN-alpha, IFN-beta, and IL-6. In contrast, poly(I:C) treatment significantly protected TLR3(-/-) mice from lethal challenge despite some deficiencies in cytokine induction. There was no indication that the lack of protection was due to the fact that TLR3-deficient mice had a reduced capacity to fight infection because they were not found to be more susceptible to PTV. We conclude that TLR3 is essential to the induction of antiviral activity elicited by poly(I:C(12)U), which does not appear to be recognized by the cytosolic sensor of poly(I:C), melanoma differentiation-associated gene-5.     

A non-neutralizing antibody broadly protects against influenza virus infection by engaging effector cells

Hemagglutinin (HA) is the immunodominant protein of the influenza virus. We previously showed that mice injected with a monoglycosylated influenza A HA (HA_mg) produced cross-strain-reactive antibodies and were better protected than mice injected with a fully glycosylated HA (HA_fg) during lethal dose challenge. We employed a single B-cell screening platform to isolate the cross-protective monoclonal antibody (mAb) 651 from mice immunized with the HA_mg of A/Brisbane/59/2007 (H1N1) influenza virus (Bris/07). The mAb 651 recognized the head domain of a broad spectrum of HAs from groups 1 and 2 influenza A viruses and offered prophylactic and therapeutic efficacy against A/California/07/2009 (H1N1) (Cal/09) and Bris/07 infections in mice. The antibody did not possess neutralizing activity; however, antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis mediated by natural killer cells and alveolar macrophages were important in the protective efficacy of mAb 651. Together, this study highlighted the significance of effector functions for non-neutralizing antibodies to exhibit protection against influenza virus infection.     

MDCK cell-cultured influenza virus vaccine protects mice from lethal challenge with different influenza viruses

Influenza epidemics are major health concern worldwide. Vaccination is the major strategy to protect the general population from a pandemic. Currently, most influenza vaccines are manufactured using chicken embroynated eggs, but this manufacturing method has potential limitations, and cell-based vaccines offer a number of advantages over the traditional method. We reported here using the scalable bioreactor to produce pandemic influenza virus vaccine in a Madin-Darby canine kidney cell culture system. In the 7.5-L bioreactor, the cell concentration reached to 3.2 × 10(6) cells/mL and the highest virus titers of 256 HAU/50 μL and 1 × 10(7) TCID50/mL. The HA concentration was found to be 11.2 μg/mL. The vaccines produced by the cell-cultured system induced neutralization antibodies, cross-reactive T-cell responses, and were protective in a mouse model against different lethal influenza virus challenge. These data indicate that microcarrier-based cell-cultured influenza virus vaccine manufacture system in scalable bioreactor could be used to produce effective pandemic influenza virus vaccines.     

Use of I125 labelled poly(I). poly(C) for determination of antibodies to double-stranded RNA by immune complex absorption to nitrocellulose filters]

125I-labeled double-stranded polyribonucleotide complex was used for detection of antibodies to double-stranded RNA in sera from people and immunized animals by the method of immune complex adsorption by the nitrocellulose filters. The technique is simple and sensitive. Antibodies to double-stranded RNA WERE detected in sera from patients with different diseases and from normal individuals. Systemic lupus erythematosus sera contain as a rule higher amounts of antibodies to double-stranded RNA. Often these antibodies were measured together with those directed towards native and denatured DNA. Anti-double-stranded RNA antibodies from sera of immunised animals and patients with systemic lupus erythematosus are highly specific.     

Oral administration of lactobacilli from human intestinal tract protects mice against influenza virus infection

Aims: Our study was conducted to evaluate the potent protective effects of oral administration of probiotic Lactobacillus strains against influenza virus (Flu) infection in a mouse model. Method and Results: Lyophilized Lactobacillus rhamnosus GG (LGG) and Lactobacillus gasseri TMC0356 (TMC0356) were orally administered to BALB/c mice for 19 days. The test mice were intranasally infected with Flu A/PR/8/34 (H1N1) on day 14, and any changes in clinical symptoms were monitored. After 6 days of infection, the mice were killed and pulmonary virus titres were determined. The clinical symptom scores of mice administered oral LGG and TMC0356 were significantly ameliorated, compared to those of the control mice (P < 0·01). The pulmonary virus titres of the mice fed LGG and TMC0356 were also significantly decreased compared to those of control mice (P < 0·05). Conclusions: These results indicate that oral administration of lactobacilli, such as LGG and TMC0356, might protect a host animal against Flu infection. Significance and Impact of the Study: These results demonstrate that oral administration of selected lactobacilli might protect host animals from Flu infection by interactions with gut immunity.     

The TLR4-TRIF pathway protects against H5N1 influenza virus infection

Prestimulation of the TLR4 pathway with lipopolysaccharide (LPS) protects mice from lethal infection with H5N1 influenza virus. Here, we reveal that the TLR4-TRIF pathway is required for this protective effect by using mice whose TLR4-related molecules were knocked out. Microarray analysis of primary mouse lung culture cells that were LPS pretreated and infected with an H5N1 virus indicated that TLR3 mRNA was upregulated. Primary lung culture cells of TLR3 knockout mice showed no response to LPS pretreatment against H5N1 virus infection, suggesting that TLR3 is also involved in the preventive effect of LPS. Our data suggest that the TLR4-TRIF axis has an important role in stimulating protective innate immunity against H5N1 influenza A virus infection and that TLR3 signaling is involved in this pathway.     

Far-infrared absorption of nucleotides and poly(I)·poly(C) RNA

We have measured the ir absorption of 5′CMP, 5′IMP, and poly(I)·poly(C) from ～25 to ～500 cm^?1. From a comparison of the data with the previously measured absorption of the corresponding nucleosides and bases we can identify several “lines” associated with the deformation of the ribose ring. Out-of-plane deformation of the bases contributes strongly to vibrations near 200 cm^?1. The same ribose vibrations observed in the nucleotides are found in poly(I)·poly(C). They sharpen with increasing water absorption. A study of the spectra of poly(I)·poly(C) as a function of the adsorbed water indicates that water does not contribute in a purely additive fashion to the polynucleotide spectrum but depends on the conformation of the helix. However, the only spectral feature that shifts drastically with conformation is near 45 cm^?1. Measurements at cryogenic temperatures indicate some sharpening of the spectrum of poly(I)·poly(C). Instead, no sharpening is observed in the spectrum of the nucleotides. Shear degradation of poly(I)·poly(C) produces significant spectral changes in the 200-cm^?1 region and sharpening of the features assigned to the low-frequency ribose-ring vibrations.