R. Garg

Enhanced immune responses and protection by vaccination with respiratory syncytial virus fusion protein formulated with CpG oligodeoxynucleotide and innate defense regulator peptide in polyphosphazene microparticles

Although respiratory syncytial virus (RSV) is the leading cause of serious respiratory tract disease in children, to date no RSV vaccine is available. To produce an effective subunit vaccine, a truncated secreted version of the F protein (ΔF) was expressed in mammalian cells, purified and shown to form trimers. The ΔF protein was then formulated with a CpG oligodeoxynucleotide (ODN) and an innate defense regulator (IDR) peptide in polyphosphazene microparticles (ΔF-MP). Mice immunized either intramuscularly (IM) or intranasally (IN) with ΔF-MP developed significantly higher levels of virus-neutralizing antibodies in the sera and lungs, as well as higher numbers of IFN-γ secreting cells than mice immunized with the ΔF protein alone. In contrast, the IM delivered ΔF induced high production of IL-5 while the IN delivered ΔF did not elicit a measurable immune response. After RSV challenge, essentially no virus and no evidence of immunopathology were detected in mice immunized with ΔF-MP regardless of the route of delivery. While the mice immunized IM with ΔF alone also showed reduced virus replication, they developed enhanced levels of pulmonary IgE, IL-4, IL-5, IL-13 and eotaxin, as well as eosinophilia after challenge. The level of protection induced by the ΔF-MP formulation was equivalent after IM and IN delivery. The efficacy and safety of the ΔF-MP formulation was confirmed in cotton rats, which also developed enhanced immune responses and were fully protected from RSV challenge after vaccination with ΔF-MP. In conclusion, formulation of recombinant ΔF with CpG ODN and IDR peptide in polyphosphazene microparticles should be considered for further evaluation as a safe and effective vaccine against RSV.

Vaccination with the RSV fusion protein formulated with a combination adjuvant induces long-lasting protective immunity.

Respiratory syncytial virus (RSV) is one of the primary causative agents of upper and lower respiratory tract infections in young children, in particular infants. Recently, we reported the protective efficacy of a RSV vaccine formulation consisting of a truncated version of the fusion (F) protein formulated with a Toll-like receptor (TLR) agonist and an immunostimulatory peptide in a carrier system (ΔF/TriAdj). To evaluate the duration of immunity induced by this vaccine candidate, we carried out long-term trials. The ΔF was formulated with triple adjuvant (TriAdj) containing either polyinosinic : polycytidylic acid (polyI : C) or cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODNs) and administered intranasally to mice. One year after the second vaccination all mice were challenged with RSV. Both ΔF/TriAdj formulations mediated the induction of high levels of IgG1, IgG2a and virus-neutralizing antibodies, and IgA in the lungs. Based on the numbers of IFN-γ- and IL-5-secreting cells in the spleen, the immune response was slightly T-helper cell type 1 (Th1)-biased. This was confirmed by the presence of F85-93-specific CD8(+) effector T cells in the lungs of both ΔF/TriAdj(polyI : C)- and ΔF/TriAdj(CpG)-immunized mice. Both ΔF/TriAdj formulations induced RSV-specific CD8(+) T cells. However, ΔF/TriAdj(polyI : C) generated significantly higher IgG affinity maturation and higher numbers of RSV-specific CD8(+) effector memory T cells in lungs and CD8(+) central memory T cells in spleen and lymph nodes than ΔF/TriAdj(CpG). After RSV challenge, no virus replication and no evidence of vaccine-induced pathology were detected in mice immunized with either of the ΔF/TriAdj formulations, demonstrating that the duration of immunity induced with these vaccines is at least one year.

Induction of mucosal immunity and protection by intranasal immunization with a respiratory syncytial virus subunit vaccine formulation.

The majority of infections, including those caused by respiratory syncytial virus (RSV), occur at mucosal surfaces. As no RSV vaccine is available our goal is to produce an effective subunit vaccine with an adjuvant suitable for mucosal delivery and cross-presentation. A truncated secreted version of the RSV fusion (ΔF) protein formulated with polyI : C, an innate defence regulator peptide and polyphosphazene, induced local and systemic immunity, including affinity maturation of RSV F-specific IgG, IgA and virus-neutralizing antibodies, and F-specific CD8(+) T-cells in the lung, when delivered intranasally. Furthermore, this ΔF protein formulation promoted the production of CD8(+) central memory T-cells in the mediastinal lymph nodes and provided protection from RSV challenge. Formulation of ΔF protein with this adjuvant combination enhanced uptake by lung dendritic cells and trafficking to the draining lymph nodes. The ΔF protein formulation was confirmed to be highly efficacious and safe in cotton rats.

The role of dendritic cells in innate and adaptive immunity to respiratory syncytial virus, and implications for vaccine development

Respiratory syncytial virus (RSV) is a common human pathogen that causes cold-like symptoms in most healthy adults and children. However, RSV often moves into the lower respiratory tract in infants and young children predisposed to respiratory illness, making it the most common cause of pediatric broncheolitis and pneumonia. The development of an appropriate balanced immune response is critical for recovery from RSV, while an unbalanced and/or excessively vigorous response may lead to immunopathogenesis. Different dendritic cell (DC) subsets influence the magnitude and quality of the host response to RSV infection, with myeloid DCs mediating and plasmacytoid DCs modulating immunopathology. Furthermore, stimulation of DCs through Toll-like receptors is essential for induction of protective immunity to RSV. These characteristics have implications for the rational design of a RSV vaccine.

The respiratory syncytial virus fusion protein formulated with a novel combination adjuvant induces balanced immune responses in lambs with maternal antibodies.

Respiratory syncytial virus (RSV) causes severe respiratory illness in infants. There are no licensed vaccines to prevent RSV infection. The neonate receives short-term protection from maternally derived antibodies, which, however, can also interfere with the active response to vaccination. A RSV vaccine consisting of a truncated version of the fusion protein formulated with polyI:C, innate defense regulator peptide and polyphosphazene (ΔF/TriAdj), was evaluated in two to three week-old lambs. When delivered intrapulmonary, ΔF/TriAdj elicited IgA production in the lung in addition to a robust systemic response similar to that induced by intramuscular immunization. To investigate potential interference by maternal antibodies, pregnant ewes were vaccinated with ΔF/TriAdj. Lambs born to RSV F-immune or non-immune ewes were then given three vaccinations with ΔF/TriAdj at 3 days, 4 weeks and 8 weeks post-birth. Lambs immunized intramuscularly with ΔF/TriAdj vaccine developed high-affinity ΔF-specific serum IgG and virus neutralizing antibodies, and displayed an increase in the frequency of IFN-γ-secreting cells by in vitro restimulated peripheral blood mononuclear cells. Maternal antibodies did not interfere with the development of an immune response to ΔF/TriAdj in the newborn lambs. These results indicate that immunization of neonates with ΔF/TriAdj is effective even in the face of maternal antibodies.

Maternal immunization with respiratory syncytial virus fusion protein formulated with a novel combination adjuvant provides protection from RSV in newborn lambs.

Respiratory syncytial virus (RSV) is the causative agent of serious upper and lower respiratory tract infections in newborns and infants. Protection from RSV is crucial for neonates, and maternal immunization is one approach that holds promise for providing immediate protection to young infants against severe RSV infection. We previously reported efficacy of a subunit vaccine consisting of the fusion (F) protein formulated with a novel adjuvant (ΔF/TriAdj) in neonates. The goal of the current study was to evaluate the ΔF/TriAdj as a maternal vaccine. Pregnant ewes were vaccinated intramuscularly with ΔF/TriAdj or PBS six weeks prior to lambing, and re-vaccinated four weeks later, which resulted in transfer of maternal antibodies (MatAbs) to the newborn lambs through the colostrum. Significantly higher levels of RSV ΔF-specific serum IgG were detected in vaccinated pregnant ewes and their lambs when compared to control animals, which revealed that MatAbs were passively transferred to the offspring. All newborn lambs were challenged with RSV at three days of age. After RSV challenge, virus production and lung pathology were significantly lower in lambs that had received passively transferred antibodies than in control animals. These results indicate that maternal immunization with ΔF/TriAdj might be an alternative, safe and effective approach to provide protection against RSV in newborn and young infants.

The bovine viral diarrhea virus E2 protein formulated with a novel adjuvant induces strong, balanced immune responses and provides protection from viral challenge in cattle.

Bovine viral diarrhea virus (BVDV) is still one of the most serious pathogens in cattle, meriting the development of improved vaccines. Recently, we developed a new adjuvant consisting of poly[di(sodium carboxylatoethylphenoxy)]-phosphazene (PCEP), either CpG ODN or poly(I:C), and an immune defense regulator (IDR) peptide. As this adjuvant has been shown to mediate the induction of robust, balanced immune responses, it was evaluated in an E2 subunit vaccine against BVDV in lambs and calves. The BVDV type 2 E2 protein was produced at high levels in a mammalian expression system and purified. When formulated with either CpG ODN or poly(I:C), together with IDR and PCEP, the E2 protein elicited high antibody titers and production of IFN-γ secreting cells in lambs. As the immune responses were stronger when poly(I:C) was used, the E2 protein with poly(I:C), IDR and PCEP was subsequently tested in cattle. Robust virus neutralizing antibodies as well as cell-mediated immune responses, including CD8(+) cytotoxic T cell (CTL) responses, were induced. The fact that CTL responses were demonstrated in calves vaccinated with an E2 protein subunit vaccine indicates that this adjuvant formulation promotes cross-presentation. Furthermore, upon challenge with a high dose of virulent BVDV-2, the vaccinated calves showed almost no temperature response, weight loss, leukopenia or virus replication, in contrast to the control animals, which had severe clinical disease. These data suggest that this E2 subunit formulation induces significant protection from BVDV-2 challenge, and thus is a promising BVDV vaccine candidate; in addition, the adjuvant platform has applications in bovine vaccines in general.

Indolamine 2,3-dioxygenase expression by monocytes and dendritic cell populations in hepatitis C patients

Dendritic cells (DCs) play an important role in the induction of the primary immune response to infection. DCs may express the tryptophan‐catabolizing enzyme indolamine2,3‐dioxygenase (IDO), which is an inducer of immune tolerance. Because there is evidence that chronic hepatitis C virus (HCV) infection leads to functional impairment of certain DC populations, we analysed IDO expression in DCs and monocytes from chronically infected and recovered HCV patients. The IDO1 and ‐2 expression was increased significantly in the monocytes of chronic HCV patients but, interestingly, not in those from recovered patients. The myeloid DCs from chronically infected HCV patients also showed enhanced IDO1 expression, while no change in either IDO1 or ‐2 was found for plasmacytoid DCs. Up‐regulation of IDO1 gene expression was confirmed by the presence of enhanced kynurenine/tryptophan ratios in the plasma from chronic HCV patients. Increased IDO1 and ‐2 expression was also observed in monocytes from healthy donors infected with an adapted mutant of the HCV JFH‐1 strain ex vivo, confirming a direct effect of HCV infection. These changes in IDO expression could be prevented by treatment with the IDO inhibitor 1‐methyl tryptophan (1‐mT). Furthermore, maturation of monocyte‐derived DCs from chronically infected HCV patients, as well as well as monocyte‐derived DCs infected ex vivo with HCV, was impaired, but this was reversed by 1‐mT treatment. This suggests that IDO inhibitors may be used to treat chronic HCV patients in vivo, in conjunction with current therapies, or to activate DCs from patients ex vivo, such that they can be administered back as a DC‐based therapeutic vaccine.

A single intranasal immunization with a subunit vaccine formulation induces higher mucosal IgA production than live respiratory syncytial virus.

Respiratory syncytial virus (RSV) causes serious respiratory illness in infants and elderly. RSV infection induces short-lived immunity, which leaves people prone to re-infection. In contrast, the RSV fusion (F) protein formulated with a novel adjuvant (∆F/TriAdj) elicits long term protective immunity. A comparison of RSV-immunized mice to mice vaccinated with a single dose of ∆F/TriAdj showed no difference in IgG1 and IgG2a production; however, local IgA secreting memory B cell development and B cell IgA production were significantly lower in RSV vaccinated mice than in ∆F/TriAdj-immunized mice. This indicates a potential reason as to why long-term immunity is not induced by RSV infection. The comparison also revealed that germinal center lymphocyte populations were higher in ∆F/TriAdj-vaccinated mice. Furthermore, ∆F/TriAdj induced higher gene expression of activation-induced cytidine deaminase (AID), as well as IL-6, IL-21, TGF-β cytokines, which are key players in IgA class switch recombination, ultimately leading to a sustained long-term memory response.

Formulation of the respiratory syncytial virus fusion protein with a polymer-based combination adjuvant promotes transient and local innate immune responses and leads to improved adaptive immunity.

Respiratory syncytial virus (RSV) causes serious upper and lower respiratory tract infections in newborns and infants. Presently, there is no licensed vaccine against RSV. We previously reported the safety and efficacy of a novel vaccine candidate (ΔF/TriAdj) in rodent and lamb models following intranasal immunization. However, the effects of the vaccine on the innate immune system in the upper and lower respiratory tracts, when delivered intranasally, have not been characterized. In the present study, we found that ΔF/TriAdj triggered transient production of chemokines, cytokines and interferons in the nasal tissues and lungs of BALB/c mice. The types of chemokines produced were consistent with the populations of immune cells recruited, i.e. dendritic cells, macrophages and neutrophils, in the nose-associated lymphoid tissue (NALT), lung and their draining lymph nodes of the ΔF/TriAdj-immunized group. In addition, ΔF/TriAdj stimulated cellular activation with generation of mucosal and systemic antibody responses, and conferred complete protection from viral infection in the lungs upon RSV challenge. The effect of ΔF/TriAdj was short-lived in the nasal tissues and more prolonged in the lungs. In addition, both innate and adaptive immune responses were lower when mice were immunized with ΔF alone. These results suggest that ΔF/TriAdj modulates the innate mucosal environment in both upper and lower respiratory tracts, which contributes to robust adaptive immune responses and long-term protective efficacy of this novel vaccine formulation.