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Respiratory syncytial virus (RSV) causes acute respiratory tract infections in infants, the elderly and immunocompromised individuals. No licensed vaccine is available against RSV. We previously reported that intranasal immunization of rodents and lambs with a RSV vaccine candidate (ΔF/TriAdj) induces protective immunity with a good safety profile. ΔF/TriAdj promoted innate immune responses in respiratory mucosal tissues in vivo, by local chemokine and cytokine production, as well as infiltration and activation of immune cells including macrophages. The macrophage is an important cell type in context of both innate and adaptive immune responses against RSV. Therefore, we characterized the effects of ΔF/TriAdj on a murine macrophage cell line, RAW264.7, and bone marrow-derived macrophages (BMMs). A gene expression study of pattern recognition receptors (PRRs) revealed induction of endosomal and cytosolic receptors in RAW264.7 cells and BMMs by ΔF/TriAdj, but no up-regulation by ΔF in PBS. As a secondary response to the PRR gene expression, induction of several chemokines and pro-inflammatory cytokines, as well as up-regulation of MHC-II and co-stimulatory immune markers, was observed. To further investigate the mechanisms involved in ΔF/TriAdj-mediated secondary responses, we used relevant signal transduction pathway inhibitors. Based on inhibition studies at both transcript and protein levels, JNK, ERK1/2, CaMKII, PI3K and JAK pathways were clearly responsible for ΔF/TriAdj-mediated chemokine and pro-inflammatory cytokine responses, while the p38 and NF-κB pathways appeared to be not or minimally involved. ΔF/TriAdj induced IFN-β, which may participate in the JAK-STAT pathway to further amplify CXCL-10 production, which was strongly up-regulated. Blocking this pathway by a JAK inhibitor almost completely abrogated CXCL-10 production and caused a significant reduction in the cell surface expression of MHC-II and co-stimulatory immune markers. These data demonstrate that ΔF/TriAdj induces multiple signaling pathways in macrophages.

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.

Bovine viral diarrhea virus (BVDV) is one of the most serious pathogens in cattle. Recently, we developed a novel adjuvant platform (TriAdj) that includes a toll-like receptor 3 agonist, poly (I:C); an innate defense regulatory peptide; and water-soluble polymer, poly[di(sodiumcarboxylatoethylphenoxy)]-phosphazene (PCEP). To develop a needle-free intradermal (ID) subunit vaccine, the BVDV type-2 E2 protein was formulated with TriAdj, and immune protection was evaluated in calves against a BVDV-2 strain. Intradermal delivery of E2/TriAdj elicited robust virus neutralizing antibodies and cell-mediated immune responses including CD4+ and CD8+ T-cell responses. The development of CD8+ T-cell responses in vaccinated calves indicates that TriAdj promotes cross-presentation. Upon challenge with virulent BVDV-2, the vaccinated calves showed no weight loss, leukopenia or virus shedding, and almost no temperature increase, in contrast to the control animals, which had severe clinical disease and shed virus for three to six days in nasal fluids and white blood cells. Intradermal vaccination was shown to attract various immune cell populations including dendritic cells, the most important antigen presenting cells. These data demonstrate that ID delivery is suitable as an administration route in cattle and that ID delivered, TriAdj-formulated E2 can protect cattle from BVDV-2.

The highly pathogenic avian influenza (HPAI) H5N1 viruses and their spillover into the human population pose substantial economic and public health threats. Although antiviral drugs have some effect in treating influenza infection, vaccination is still the most effective intervention to prevent possible pandemic outbreaks. We have developed a novel H5 influenza vaccine to improve the world's pandemic preparedness. We produced a hemagglutinin (HA) of HPAI H5N1 virus A/Alberta/01/2014 (AB14) using both mammalian (m) and bacterial (b) expression systems. The purified recombinant proteins were formulated with a proprietary adjuvant (TriAdj) and their efficacy as vaccine candidates was evaluated in mice. Intramuscular delivery of two doses of TriAdj formulated mammalian expressed HA (m-HA/TriAdj) was shown to provide full protection against a lethal challenge of AB14 in mice. In contrast, bacterially expressed HA with TriAdj (b-HA/TriAdj), b-HA without adjuvant, and m-HA without adjuvant resulted in no protection in immunized mice. Furthermore, m-HA/TriAdj elicited significantly higher levels of balanced Th1 and Th2 responses and neutralizing antibody titres. All the mice in the m-HA/TriAdj group survived a lethal AB14 H5N1 challenge and showed no signs of disease or infection as demonstrated by no loss of body weight or detectable virus in the lungs. Our results suggest that m-HA formulated with TriAdj has potential to protect against pandemic H5N1 in the event of its cross over to the human host.

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.

Adjuvants are crucial components of many vaccines. They are used to improve the immunogenicity of vaccines with the aim of conferring long-term protection, to enhance the efficacy of vaccines in newborns, elderly or immunocompromised persons, and to reduce the amount of antigen or the number of doses required to elicit effective immunity. Novel combination adjuvants have been tested in both candidate animals and humans vaccines and have generated encouraging results. Recently, we developed a combination adjuvant platform (TriAdj) comprising of three components, namely a TLR agonist, either polyI:C or CpG oligodeoxynucleotides (ODN), host defense peptide and polyphosphazene. This adjuvant platform is stable and highly effective in a wide range of animal and human vaccines tested in mice, cotton rats, pigs, sheep, and koalas. TriAdj with various vaccines antigens induced effective long-term humoral and cellular immunity. Moreover, the adjuvant platform is suitable for maternal immunization and highly effective in neonates even in the presence of maternal antibodies. This novel vaccine platform, offers excellent opportunity for use in present and future generations of vaccines against multiple infectious agents and targets challenging populations.

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.

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.

Bovine viral diarrhea virus (BVDV) is one of the major pathogens in cattle. In this study, newborn calves with maternal antibodies were vaccinated with a BVDV DNA vaccine, either by conventional intramuscular (IM) injection or with the TriGrid™ Delivery System for IM delivery (TDS-IM). The calves vaccinated with the TDS-IM developed more rapidly and effectively BVDV-specific humoral and cell-mediated immune responses in the presence of maternal antibodies. Overall, the immune responses induced by delivery with the TDS-IM remained stronger than those elicited by conventional IM injection of the BVDV DNA vaccine. Accordingly, electroporation-mediated delivery of the BVDV DNA vaccine resulted in close to complete protection from clinical signs of disease, while conventional IM administration did not fully prevent morbidity and mortality following challenge with BVDV-2. These results demonstrate the TDS-IM to be effective as a delivery system for a BVDV DNA vaccine in newborn calves in the presence of maternal antibodies, which supports the potential of electroporation as a delivery method for prophylactic DNA vaccines.

Host defence peptides (HDPs) have a variety of potential therapeutic applications, including as vaccine adjuvants, energizing efforts for modification strategies to address their toxicity and instability. Here we compare l, d and RI-Bac2A as vaccine adjuvants. d and RI-Bac2A are equally resistant to proteolytic degradation with no increases in toxicity, however, only RI-Bac2A maintains adjuvant activity of the natural peptide through conserved induction of a Th2 immune response. As HDPs potentiate the adjuvant activity of CpG ODNs, the isomers were also evaluated as co-adjuvants. l-Bac2A has no significant co-adjuvant activity while CpG/RI-Bac2A induces antibody titres significantly higher than CpG (P0.01), CpG/l-Bac2A (P0.01) or CpG/d-Bac2A (P0.01). None of the isomers influence ODN duration or distribution but l and RI-Bac2A promote ODN uptake into B cells and antigen presenting cells. The enhanced adjuvant and co-adjuvant of RI-Bac2A is hypothesized to result from an undefined combination of increased stability and retained biological activity supporting application of retro-inversion to this, and potentially other HDPs.

Vaccines consisting of subunit or protein antigens are less immunogenic than traditional vaccines, and therefore require formulation with an adjuvant. Conventional adjuvants, however, often cause undesirable injection site reactions and Th2-biased immune responses. Therefore, novel vaccine adjuvants which can safely enhance and selectively bias the resulting immune response are required. Here the adjuvant combination of CpG ODN, indolicidin and polyphosphazene (CpG+indol+PP) was evaluated for its ability to enhance and modulate the immune response when formulated with the antigen hen egg lysozyme (HEL). Cattle immunized with HEL co-adjuvanted with CpG+indol+PP developed higher antigen-specific humoral responses, and long-lasting cell-mediated immune responses, as evidenced by elevated levels of IFN-gamma secretion by re-stimulated PBMCs, that were superior even to EMULSIGEN((R)), an oil-in-water based adjuvant that was used as positive control. Physical characterization of the vaccines indicated that formulation of HEL with CpG+indol+PP resulted in the formation of antigen-adjuvant complexes, which may have contributed to their enhanced immunogenicity. Furthermore, the addition of polyphosphazene to CpG ODN and indolicidin dose-dependently enhanced the secretion of the cytokines IFN-alpha, TNF-alpha and IFN-gammain vitro, indicating that polyphosphazene can also synergize with CpG ODN and indolicidin to stimulate innate immune responses.

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 TriGrid™ 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.

Type 2 bovine viral diarrhea virus (BVDV) has become increasingly prevalent worldwide, and currently the ratio of type 2 to type 1 strains in the USA approaches 50%. Although there is cross-reactivity between BVDV type 1 and type 2 strains, BVDV1 vaccine strains poorly protect from type 2 infection, so vaccines against BVDV should contain antigens from both BVDV types. Previously we demonstrated efficacy of a BVDV1 E2 DNA vaccine, and in this study we optimized a BVDV2 E2 DNA vaccine. Furthermore, as an approach to vaccinate with a DNA vaccine against both BVDV types, we compared two strategies, mixing of plasmids encoding type 1 and type 2 E2, and co-expression of type 1 and type 2 E2 from one plasmid with an internal ribosomal entry site (IRES). An evaluation of the IRES-containing plasmids demonstrated that the C-terminally expressed protein is produced at lower levels and induces weaker immune responses than the N-terminally expressed protein, regardless of the position of the type 1 and type 2 E2 genes. In contrast, when both plasmids encoding type 1 and type 2 E2 were administered to mice, the immune responses were similar to those induced by the individual plasmids. Thus, a mixture of plasmids encoding type 1 and type 2 E2 could be a potential DNA vaccine candidate against both BVDV1 and BVDV2.

In this study, we tested the hypothesis that adoptive transfer of dendritic cells (DCs) transfected ex vivo with mRNA encoding hepatitis C virus (HCV) NS3/4A would initiate potent HCV-specific protective immune responses in vivo. Murine DCs were transfected with NS3/4A mRNA or eGFP mRNA using either electroporation or Transmessenger Transfection Reagent and then used for adoptive transfer. Electroporation resulted in higher transfection efficiency but lower levels of eGFP and NS3/4A expression when compared to transfection with Transmessenger. The murine NS3/4A mRNA-transfected DCs were functional in T cell activation in vitro. Adoptive transfer of NS3/4A mRNA-transfected DCs resulted in migration to regional lymph nodes, strong cellular immune responses and protection from challenge with vaccinia virus expressing NS3/NS4/NS5 in mice. Furthermore, although Transmessenger mediated transfection was less efficient than electroporation in terms of number of transfected cells, the DCs transfected with NS3/4A mRNA and Transmessenger expressed higher levels of protein and induced stronger immune responses and protection than DCs transfected with NS3/4A mRNA by electroporation. Since no study has explored the in vivo efficacy of mRNA-transfected DC-mediated vaccination against viral diseases, including hepatitis C, our study provided a novel vaccination strategy against hepatitis C as well as other pathogens.

Bovine herpesvirus 1 (BHV-1) causes respiratory and genital infections in cattle. Previously we demonstrated that a DNA vaccine encoding a truncated, secreted form of BHV-1 glycoprotein B (tgB) induces cytotoxic T lymphocyte (CTL) responses in C3H mice. In this study we investigated the potential of interleukin 12 (IL-12) to further enhance the CTL response. C3H mice were immunized with a plasmid encoding tgB or with plasmids encoding tgB and murine IL-12. When the plasmid encoding tgB was delivered intramuscularly or epidermally by a gene gun, co-administration with IL-12 plasmid stimulated the synthesis of more IgG2a, the production of higher levels of IFN-gamma, and more effective killing by CTLs. In contrast, after intradermal delivery no effect of co-administration of IL-12 encoding plasmid was observed. Further investigation suggested that antigen and IL-12 need to be expressed in the draining lymph nodes, where IL-12 can have a direct effect on T cells.

The efficacy of oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG ODN) as an adjuvant for rabbits was assessed alone and in combination with aluminum hydroxide (CpG/alum). The CpG/alum combination elicited a greater immune response to several antigens compared to Freund's adjuvant. A non-CpG/alum combination did not have the same effects as CpG/alum suggesting that the adjuvanticity was related to the CpG motifs. In addition, we formulated one of the antigens with combinations of CpG ODN and 30 or 10% Emulsigen (Em) [CpG/Em (30%) and CpG/Em (10%)]. Both CpG/Em (30%) and CpG/Em (10%) were more effective than Em, and equivalent to CpG/alum. The CpG/Em (10%) combination caused minimal tissue damage. Our results demonstrate that the addition of CpG ODN to aluminum hydroxide or to 10% Em significantly improves the efficiency of these adjuvants, without enhancing tissue reaction.

The adjuvanticity of a synthetic oligodeoxynucleotide containing unmethylated CpG motifs (CpG ODN) was determined in cattle. Calves were immunized with a truncated secreted version of glycoprotein D (tgD) of bovine herpes virus-1 (BHV-1) formulated with alum, CpG ODN, or a combination of both. BHV-1 tgD formulated with CpG ODN or with alum and CpG ODN induced a stronger and more balanced immune response than tgD in alum. This level of immunity was of sufficient magnitude to minimize weight loss and significantly reduce the duration of virus shedding after intranasal viral challenge. Local tissue reactions generated by CpG ODN were very mild and transient, whereas reactions induced by alum or a combination of CpG ODN and alum were moderate in severity and duration. These data demonstrate that CpG ODN causes minimal injection site reactions and yet acts as an effective adjuvant in cattle.

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.

Glycoprotein D (gD) is essential for attachment and penetration of Bovine herpesvirus 5 (BoHV-5) into permissive cells, and is a major target of the host immune system, inducing strong humoral and cellular immune responses. The aim of this study was to evaluate in mice the immunogenicity of recombinant BoHV-5 gD (rgD5) expressed in Pichia pastoris. Vaccines formulated with rgD5 alone or adjuvanted with Montanide 50 ISA V2; Emulsigen or Emulsigen-DDA was administered intramuscularly or subcutaneously. Almost all formulations stimulated a humoral immune response after the first inoculation. The only exception was observed when the rgD5 was administered subcutaneously without adjuvant, in this case, the antibodies were observed after three doses. Higher titers of neutralizing antibodies were obtained with the three oil-based adjuvant formulations when compared to non-adjuvanted vaccine formulations. The rgD5 vaccine stimulated high mRNA expression levels of Th1 (INF-γ) and pro-inflammatory cytokines (IL-17, GM-CSF). The results demonstrated that the recombinant gD from BoHV-5 conserved important epitopes for viral neutralization from native BoHV-5 gD and was able to elicit mixed Th1/Th2 immune response in mice.

Activation of different T-helper (Th) responses following immunisation has profound and specific influences on the development of the immune response and on the ability of a vaccine to confer protection. Since cytokines are capable of influencing the stimulation of distinct T-cell responses, their encapsulation in vaccines should modulate antigen-specific immune responses. Unfortunately, the use of cytokines in vivo is hampered by their rapid clearance and inactivation. One possible solution to this problem is the use of liposomes to entrap both cytokines and antigen. This approach will not only protect the cytokine but will also deliver the two components simultaneously to the same site. The authors examined, therefore, the immune responses elicited by systemic immunisation of mice with liposome formulations containing a truncated form of bovine herpesvirus type-1 glycoprotein D (tgD) together with IL-4 or IL-12. Subcutaneous immunisation with liposomes containing tgD and IL-12 significantly enhanced the induction of antigen-specific cellular and humoral immune responses. These responses were characterised by an increase in IFN-γ secreting cells and the induction of tgD-specific IgG2a antibodies. In contrast, encapsulation of IL-4 into tgD-liposomes did not enhance the humoral immune response to gD but significantly influenced the development of antigen-specific IL-4 secreting cells. Our results indicated that encapsulation of IL-12 into the liposomes was necessary for the systemic adjuvant effect and demonstrated the feasibility of using liposome technology and cytokines to manipulate the development of different antigen-specific Th subsets in vivo.

We immunized cattle, the natural host for bovine herpesvirus 1 (BHV-1), with a polynucleotide vaccine encoding BHV-1 glycoprotein D. These cattle trials clearly indicate that large species can be immunized with polynucleotide vaccines. Recently, using a murine model, we demonstrated that: the cellular compartment to which the expressed antigen is delivered determines the type of immune response (type 1 or type 2), and that the magnitude and direction of the immune response can be modulated by coadministration of plasmid encoded cytokines and antigen. Finally, we demonstrated that immunization of mice with a polynucleotide vaccine encoding BHV-1 gD could circumvent preexisting passively transferred, gD specific, polyclonal antisera and lead to the development of an active immune response.

The efficacy of two experimental subunit gD vaccines and two commercial whole virus vaccines was determined in a bovine herpesvirus-1 (BHV1) challenge trial. Full-length gD and a truncated, secreted form of gD (tgD) were produced using a vaccinia virus expression system and purified by affinity chromatography. Comparison of these forms of gD did not reveal significant structural or antigenic differences. Calves immunized with gD or tgD in avridine developed significantly (P

The development of vaccines for infants and young children requires the use of animal models at various stages of preclinical development. Animal models are being used to assess the quantity and quality of the immune response, onset and duration of the response, induction of systemic versus local immunity, protection against challenge infection for the assessment of vaccine efficacy, as well as safety and toxicity of the vaccine formulation itself. A variety of animal models are available, each with its own specific advantages and disadvantages. Here, we review the most common animal models for preclinical vaccine development for human infants.

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.