Maternal vaccination with a novel chimeric glycoprotein formulated with a polymer-based adjuvant provides protection from human parainfluenza virus type 3 in newborn lambs.

Human parainfluenza virus 3 (PIV3) and respiratory syncytial virus (RSV) are major causative agents of serious respiratory tract illness in newborns and infants. Maternal vaccination could be a promising approach to provide immediate protection against severe PIV3 and RSV infection in young infants. Previously, we demonstrated that maternal immunization with a subunit vaccine consisting of the RSV fusion (F) protein formulated with TriAdj, an adjuvant consisting of poly(I:C), immune defense regulatory peptide and polyphosphazene, protects newborn lambs from RSV. In the present study we evaluated the protective efficacy of a novel bivalent RSV-PIV3 vaccine candidate, FRipScHN/TriAdj, as a maternal vaccine against PIV3 infection in a neonatal lamb model. This vaccine consists of the pre-fusion form of the RSV F protein linked to the haemagglutinin-neuraminidase (HN) of PIV3, formulated with TriAdj. First, we successfully established PIV3 infection in neonatal lambs. Lambs infected with human PIV3 showed gross pathology, bronchointerstitial pneumonia and viral replication in the lungs. Subsequently, ewes were immunized with FRipScHN/TriAdj. RSV FRipSc- and PIV3 HN-specific antibodies with virus-neutralizing activity were detected in both the serum and the colostrum of the vaccinated ewes. The newborn lambs had RSV- and PIV3- neutralizing antibodies in their serum, which demonstrates that maternal antibodies were transferred to the neonates. At three days of age, the newborn lambs received an intrapulmonary challenge with PIV3. The lung pathology and virus production were significantly reduced in lambs that had received PIV3-specific maternal antibodies compared to lambs born to non-vaccinated ewes. These results suggest that maternal vaccination with a bivalent FRipScHN/TriAdj vaccine might be an effective method to provide protection against both PIV3 and RSV in neonates.

A chimeric glycoprotein formulated with a combination adjuvant induces protective immunity against both human respiratory syncytial virus and parainfluenza virus type 3.

Human respiratory syncytial virus (RSV) and parainfluenza virus type 3 (PIV3) are major causes of serious lower respiratory tract disease in infants. Currently there is no licensed vaccine against RSV or PIV3. To make an effective bivalent subunit vaccine, a chimeric truncated FRHN protein containing the N-terminal ectodomain of the RSV fusion (F) protein linked to the C-terminal ectodomain of the PIV3 haemagglutinin-neuraminidase (HN) protein was produced in HEK293T cells. Mice, cotton rats and hamsters were immunized intramuscularly (IM) with both RSV F and PIV3 HN (FR+HN) or FRHN, formulated with TriAdj, which consists of poly(I:C), innate defense regulator peptide and poly[di(sodium carboxylatoethylphenoxy)]-phosphazene. Both formulations were immunogenic and elicited full protection from RSV; however, animals vaccinated with FRHN/TriAdj were significantly better protected from PIV3 than animals vaccinated with FR+HN/TriAdj. To develop a potentially more effective subunit vaccine, a chimeric glycoprotein (FRipScHN), encoding the RSV F ectodomain stabilized in the pre-fusion form linked to PIV3 HN was generated. Intramuscular vaccination with FRipScHN/TriAdj induced virus neutralizing antibodies followed by complete protection from RSV and PIV3 replication in the lungs of challenged cotton rats. Furthermore, intranasal vaccination with FRipScHN/TriAdj significantly reduced both RSV and PIV3 replication in cotton rats. Mucosal immunization with FRipScHN/TriAdj also elicited strong antigen-specific mucosal and systemic immune responses in a lamb model. In conclusion, the chimeric FRipScHN protein combined with TriAdj has potential for development of a safe, effective, bivalent vaccine against both RSV and PIV3.

Vaccination with a human parainfluenza virus type 3 chimeric FHN glycoprotein formulated with a combination adjuvant induces protective immunity.

Human parainfluenza virus type 3 (PIV3) is a major cause of lower respiratory disease i.e. bronchitis, bronchiolitis or pneumonia, in infants and young children. Presently there is no licensed vaccine against PIV3. To produce an effective subunit vaccine, a chimeric FHN glycoprotein consisting of the N-terminal ectodomain of the fusion (F) protein linked to the haemagglutinin-neuraminidase (HN) protein without transmembrane domain, and secreted forms of the individual F and HN glycoproteins, were expressed in mammalian cells and purified. Mice and cotton rats were immunized intramuscularly (IM) with FHN or both F and HN proteins (F + HN), formulated with poly(I:C) and an innate defense regulator peptide in polyphosphazene (TriAdj). Significantly higher levels of systemic virus-neutralizing antibodies were observed in mice and cotton rats immunized with FHN/TriAdj when compared to animals immunized with the combination of F and HN proteins (F + HN/TriAdj). As PIV3 is a pneumotropic virus, another goal is to produce an effective mucosal subunit vaccine. Intranasal (IN) administration with FHN/TriAdj resulted in mucosal IgA production in the lung and virus neutralizing antibodies in the sera. After PIV3 challenge no virus was detected in cotton rats immunized with FHN/TriAdj regardless of the route of delivery. Protective immunity against PIV3 was also induced by FHN/TriAdj in hamsters. In conclusion, the FHN protein formulated with TriAdj has potential for development of a safe and effective vaccine against PIV3.

Intranasal immunization with a single dose of the fusion protein formulated with a combination adjuvant induces long-term protective immunity against respiratory syncytial virus.

Respiratory syncytial virus (RSV) is the most common cause of respiratory tract infections in both children and elderly people. In this study we evaluated the short- and long-term protective efficacy of a single intranasal (IN) immunization with a RSV vaccine formulation consisting of a codon-optimized fusion (F) protein formulated with poly(I:C), an innate defense regulator peptide and a polyphosphazene (ΔF/TriAdj). This vaccine induced strong systemic and local immune responses, including RSV F-specific IgG1 and IgG2a, SIgA and virus neutralizing antibodies in mice. Furthermore, ΔF/TriAdj promoted production of IFN-γ-secreting T cells and RSV F85-93-specific CD8+ effector T cells. After RSV challenge, no virus was recovered from the lungs of the vaccinated mice. To evaluate the duration of immunity induced by a single IN vaccination, mice were again immunized once with ΔF/TriAdj and challenged with RSV five months later. High levels of IgG1, IgG2a and virus neutralizing antibodies were detected in the ΔF/TriAdj-vaccinated animals. Moreover, this vaccine formulation induced robust local SIgA production and IgA-secreting memory B cell development, and conferred complete protection against subsequent RSV challenge. In conclusion, a single IN vaccination with RSV ΔF protein formulated with TriAdj induced robust, long-term protective immune responses against RSV infection.

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 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.

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.

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.

Early postnatal immunisation against gonadotrophin-releasing hormone induces a high but differential immune response in heifer calves.

The aim of this study was to evaluate endocrinological and immunological effects of early postnatal immunisation against gonadotrophin-releasing hormone (GnRH) in heifer calves, as similar treatment in sheep provokes long-term immunocastration. Heifer calves were injected with either a construct of GnRH - bovine herpes virus 1 glycoprotein D (BHV1 gD; n=9) or saline (n=9) at 2, 6 and 13.5 weeks of age. Antibody (GnRH and carrier) and endocrine responses to immunisation were measured twice monthly (FSH and progesterone) or during intensive sampling regimes (LH). Early postnatal immunisation against GnRH induced a high, but variable, antibody response against both GnRH and carrier. Based on antibody responses, animals were divided into high-titre (HT, n=5) and low-titre (LT, n=4). Occurring mainly in HT, a further peak in anti-GnRH antibodies, stimulated independently of the carrier, was observed at 23 weeks of age, with antibody titres ≥ 10% binding for ≈ 9 weeks post-peak. Conversely immunisation had only temporary, reversible effects on reproductive function, not affecting age at puberty. We hypothesise that the newly generated antibody measured 10 weeks after the final immunisation resulted from antigenic stimulation and immunological memory cell activation to an endogenous GnRH release. This outcome offers an opportunity for further manipulation of reproductive function based on modulation of GnRH secretion and activity where long-term immunological memory may contribute to durable endocrine effects.

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.

Human dendritic cells expressing hepatitis C virus core protein display transcriptional and functional changes consistent with maturation.

Hepatitis C virus (HCV) causes a chronic liver infection, which may result in cirrhosis and hepatocellular carcinoma. Impairment of the maturation process in dendritic cells (DCs) may be one of the mechanisms responsible for immune evasion of HCV. The core and NS3 proteins are among the most conserved HCV proteins and play a key role in viral clearance. To evaluate the effects of these proteins on DCs, monocyte-derived immature DCs (iDCs) were transfected with in vitro transcribed (IVT) HCV core or NS3 RNA and treated with maturation factors. Neither core nor NS3 had an inhibitory effect on DC maturation; however, transfection of iDCs with IVT core RNA appeared to result in changes compatible with maturation. To investigate this in more detail, the transcriptional profiles of iDCs transfected with IVT core, NS3 or green fluorescent protein (GFP) RNA were examined using a DC-specific membrane array. Of the 288 genes on the array, 46 genes were distinctively up- or down-regulated by transfection with IVT core RNA in comparison with NS3 or GFP RNA treatments. Forty-two of these genes are involved in DC maturation. The effects of core on maturation of iDCs were confirmed with a significant increase in surface expression of CD83 and HLA-DR, a reduction of phagocytosis, as well as an increase in proliferation and IFN-γ secretion by T cells in a mixed lymphocyte reaction assay. These results show that HCV core does not have an inhibitory effect on human DC maturation, but could be a target for the immune system.

The novel adjuvant combination of CpG ODN, indolicidin and polyphosphazene induces potent antibody- and cell-mediated immune responses in mice.

The need to enhance the immunogenicity of purified subunit antigens and modulate resulting immune responses has prompted the development of new adjuvants. Here, the ability of CpG oligodeoxynucleotides (ODN), a bovine host defence peptide indolicidin, and polyphosphazene to synergistically combine and enhance innate and adaptive immune responses was examined in mice. In vitro, the adjuvant combination of CpG ODN, indolicidin and polyphosphazene (CpG/indol/PP) enhanced the secretion of TNF-alpha, IL-12p40, and IL-6 by bone marrow-derived DCs (BMDCs) when compared to the individual components. When co-formulated with ovalbumin (OVA), CpG/indol/PP formed antigen-adjuvant complexes, and enhanced antibody and cell-mediated responses in mice, via both MHC I and II pathways, promoting a more balanced antibody-mediated and type 1-biased cell-mediated immune response. Furthermore, substitution of the proline residues of indolicidin with arginine increased the synergistic adjuvant effect of the peptide, and induced significantly higher IgG1 and IgG2a titers and IFN-gamma secretion, as well as increased uptake by antigen presenting cells. These results clearly demonstrate that the use of a combination of CpG ODN, indolicidin, and polyphosphazene as adjuvant can significantly enhance an antigen-specific immune response.

Formulation of bovine respiratory syncytial virus fusion protein with CpG oligodeoxynucleotide, cationic host defence peptide and polyphosphazene enhances humoral and cellular responses and induces a protective type 1 immune response in mice.

Respiratory syncytial virus (RSV) is the leading cause of serious respiratory tract disease in children and calves; however, RSV vaccine development has been slow due to early observations that formalin-inactivated vaccines induced Th2-type immune responses and led to disease enhancement upon subsequent exposure. Hence, there is a need for novel adjuvants that will promote a protective Th1-type or balanced immune response against RSV. CpG oligodeoxynucleotides (ODNs), indolicidin, and polyphosphazene were examined for their ability to enhance antigen-specific immune responses and influence the Th-bias when co-formulated with a recombinant truncated bovine RSV (BRSV) fusion protein (DeltaF). Mice immunized with DeltaF co-formulated with CpG ODN, indolicidin, and polyphosphazene (DeltaF/CpG/indol/PP) developed higher levels of DeltaF-specific serum IgG, IgG1 and IgG2a antibodies when compared with DeltaF alone, and displayed an increase in the frequency of gamma interferon-secreting cells and decreased interleukin (IL)-5 production by in vitro restimulated splenocytes, characteristic of a Th1 immune response. These results were observed in both C57BL/6 and BALB/c strains of mice. When evaluated in a BRSV challenge model, mice immunized with DeltaF/CpG/indol/PP developed significantly higher levels of BRSV-neutralizing serum antibodies than mice immunized with the DeltaF protein alone, and displayed significantly less pulmonary IL-4, IL-5, IL-13 and eotaxin and reduced eosinophilia after challenge. These results suggest that co-formulation of DeltaF with CpG ODN, host defence peptide and polyphosphazene may result in a safe and effective vaccine for the prevention of BRSV and may have implications for the development of novel human RSV vaccines.

Electroporation-based DNA transfer enhances gene expression and immune responses to DNA vaccines in cattle.

Despite the potential of DNA vaccines to induce strong, balanced immune responses in small experimental species, the immune responses to DNA immunization in larger species have generally been moderate and inconsistent. In this study, the TriGridtrade mark Delivery System (TDS), an electroporation-based DNA delivery platform, was evaluated for administration of DNA vaccines to calves. When compared to conventional intramuscular delivery, TDS-based delivery markedly and consistently enhanced gene expression from a plasmid encoding a reporter gene, secreted alkaline phosphatase, and improved cell-mediated and humoral immune responses to a plasmid encoding a model antigen, hepatitis B surface antigen. Importantly, the TDS-based procedure was well tolerated by the calves, which did not need to be anesthetized or sedated. These results suggest that the TDS is a useful delivery method for DNA vaccines in cattle.

Strategies for induction of protective immunity to bovine herpesvirus-1 in newborn calves with maternal antibodies.

The objective of this study was to evaluate Th1 promoting strategies for vaccination of neonates against bovine herpesvirus-1 (BHV-1). A plasmid encoding a secreted truncated version of glycoprotein D (tgD) and tgD protein formulated with CpG oligodeoxynucleotide (ODN) effectively primed the immune system of newborn lambs, whereas without CpG ODN the tgD protein was less effective. Furthermore, a heterologous DNA prime-protein/CpG boost induced stronger and more balanced immune responses than either the DNA vaccine or a protein/CpG prime-DNA boost. Three of these strategies were compared as an approach to induce protective immunity in newborn calves with BHV-1-specific maternal antibodies. Whereas the DNA vaccine induced minimal protection, the DNA prime-protein boost resulted in reduced temperature response, weight loss and virus shedding in comparison to the placebo group. Close to complete protection against BHV-1 challenge was elicited in the calves immunized with the protein/CpG formulation, as these animals lost very little weight, had only slightly elevated temperatures and shed almost no virus.

DNA prime protein boost strategies protect cattle from bovine viral diarrhea virus type 2 challenge.

At present, infections with bovine viral diarrhea virus (BVDV) type 2 occur nearly as frequently as those with BVDV type 1, so development of vaccines that protect cattle from both type 1 and type 2 BVDV has become critical. In this study, we compared various DNA prime-protein boost vaccination strategies to protect cattle from challenge with BVDV-2 using the major protective antigen of BVDV, glycoprotein E2. Calves were immunized with a plasmid encoding either type 1 E2 (E2.1) or type 2 E2 (E2.2) or with both plasmids (E2.1+E2.2). This was followed by a heterologous boost with E2.1, E2.2 or E2.1 and E2.2 protein formulated with Emulsigen and a CpG oligodeoxynucleotide. Subsequently, the calves were challenged with BVDV-2 strain 1373. All vaccinated calves developed both humoral and cell-mediated immune responses, including virus-neutralizing antibodies and IFN-gamma-secreting cells in the peripheral blood. Depletion studies showed that CD4+ T cells were responsible for IFN-gamma production. Furthermore, the calves vaccinated with either the E2.2 or the E2.1+E2.2 vaccines were very well protected from challenge with BVDV-2, having little leukopenia and showing no weight loss or temperature response. In addition, the animals vaccinated with the E2.1 vaccine were partially protected, so there was a certain level of cross-protection. These data demonstrate that a vaccination strategy consisting of priming with E2.2 or E2.1+E2.2 DNA and boosting with E2.2 or E2.1+E2.2 protein fully protects cattle from BVDV-2 challenge.

Strategies for loading dendritic cells with hepatitis C NS5a antigen and inducing protective immunity.

Dendritic cell (DC)-based vaccination strategies are promising for the treatment of cancers and infectious diseases including hepatitis C virus (HCV). As the induction of T cell-mediated immune responses by DC vaccination is highly dependent on efficient antigen loading of the DCs, the purpose of this study was to identify an optimal nonviral DC loading strategy for HCV NS5a. Furthermore, the efficacy of immunization with the NS5a-loaded DCs in comparison to plasmid encoding NS5a and NS5a protein was evaluated. Transfection of DCs with mRNA was most efficient with close to 100% of DCs expressing NS5a, whereas approximately 10% of protein-pulsed DCs and <1% of plasmid-transfected DCs expressed NS5a, suggesting remarkably different loading efficiencies. Vaccination of mice with NS5a mRNA-transfected DCs or NS5a protein-pulsed DCs resulted in significantly stronger CD4(+) and CD8(+) T-cell responses and protection from challenge with vaccinia virus expressing NS3/NS4/NS5, in comparison to vaccination with NS5a DNA-transfected DCs, plasmid encoding NS5 or rNS5a protein formulated with alum. Furthermore, vaccination with NS5a mRNA-transfected DCs was superior to vaccination with rNS5a-pulsed DCs. These data have important clinical implications, with mRNA-transfected DCs providing a safe and effective vaccination strategy against hepatitis C and possibly other pathogens.

Immunopathology of RSV infection: prospects for developing vaccines without this complication.

Respiratory syncytial virus is the most important cause of lower respiratory tract infection in infants and young children. RSV clinical disease varies from rhinitis and otitis media to bronchiolitis and pneumonia. An increased incidence of asthma later in life has been associated with the more severe lower respiratory tract infections. Despite its importance as a pathogen, there is no licensed vaccine against RSV. This is due to a number of factors complicating the development of an effective and safe vaccine. The immunity to natural RSV infection is incomplete as re-infections occur in all age groups, which makes it challenging to design a protective vaccine. Second, the primary target population is the newborn infant, which has a relatively immature immune system and maternal antibodies that can interfere with vaccination. Finally, some vaccines have resulted in a predisposition for exacerbated pulmonary disease in infants, which was attributed to an imbalanced Th2-biased immune response, although the exact cause has not been elucidated. This makes it difficult to proceed with vaccine testing in infants. It is likely that an effective and safe vaccine needs to elicit a balanced immune response, including RSV-specific neutralising antibodies, CD8 T-cells, Th1/Th2 CD4 T-cells and preferably secretory IgA. Subunit vaccines formulated with appropriate adjuvants may be adequate for previously exposed individuals. However, intranasally delivered genetically engineered attenuated or vectored vaccines are currently most promising for newborns, as they are expected to induce a balanced immune response similar to that elicited to natural infection and not be subject to interference from maternal antibodies. Maternal vaccination may be the optimal strategy to protect the very young infants.

Formulation with CpG ODN enhances antibody responses to an equine influenza virus vaccine.

Previous studies have shown that protection against equine influenza virus (EIV) is partially mediated by virus-specific IgGa and IgGb. In this study we tested whether addition of a CpG ODN formulation to a commercial killed virus vaccine would enhance EIV-specific IgGa and IgGb antibody responses, and improve protection against an experimental EIV challenge. Thirty naïve horses were assigned to one of three groups and vaccinated as follows: 10 were given vaccine (Encevac TC4, Intervet Inc.) alone, 10 were given vaccine plus 0.25 mg CpG ODN 2007 formulated with 30% Emulsigen (CpG/Em), and 10 controls were given saline. All horses were challenged with live virus 12 weeks after the final vaccination. Antibody responses were tested by single radial hemolysis (SRH) and ELISA, and protection was evaluated by determination of temperature, coughing, and clinical scores. Killed virus vaccine combined with CpG/Em induced significantly greater serologic responses than did the vaccine alone. All antibody isotypes tested increased after the addition of CpG/Em, although no shift in relative antibody isotypes concentrations was detected. Vaccination significantly improved protection against challenge but the differences between the two vaccine groups were not statistically significant. This study is the first demonstration that CpG/Em enhances antigen-specific antibody responses in horses and supports its potential to be used as an adjuvant for vaccines against equine infections.