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Introduction: Adjuvants form an integral component in most of the inactivated and subunit vaccine formulations. Careful and proper selection of adjuvants helps in promoting appropriate immune responses against target pathogens at both innate and adaptive levels such that protective immunity can be elicited. Areas covered: Herein, we describe the recent progress in our understanding of the mode of action of adjuvants that are licensed for use in human vaccines or in clinical or pre-clinical stages at both innate and adaptive levels. Different pathogens have distinct characteristics, which require the host to mount an appropriate immune response against them. Adjuvants can be selected to elicit a tailor-made immune response to specific pathogens based on their unique properties. Identification of biomarkers of adjuvanticity for several candidate vaccines using omics-based technologies can unravel the mechanism of action of modern and experimental adjuvants. Expert opinion: Adjuvant technology has been revolutionized over the last two decades. In-depth understanding of the role of adjuvants in activating the innate immune system, combined with systems vaccinology approaches, have led to the development of next-generation, novel adjuvants that can be used in vaccines against challenging pathogens and in specific target populations.

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.

CpG oligodeoxynucleotides (CpG ODNs) which can induce innate immune responses and promote adaptive immune responses, are powerful tools in defeating diseases. Here, a novel chitosan nanoparticle (CS-NPs) based on host-guest interaction has been designed for encapsulation and delivery of CpG ODNs for the first time. The CS-NPs exhibited high encapsulation efficiency (98.3%) of CpG ODNs and remained stable in storage under room temperature for at least 7 days. CS-NPs can also prevent CpG ODN diffusion at pH 7. The results of confocal laser scanning microscope images and flow cytometry show that CS-NPs can also be efficiently delivered into living cells. Furthermore, CpG@CS-NPs can increase the immunostimulatory activity of CpG ODNs. Raw 264.7 cells treated with CpG@CS-NPs demonstrated upregulation of both TNF-α and IL-6 cytokines by 13% and 40%, respectively. The newly developed CpG@CS-NPs were thus identified as an efficient system to deliver CpG-ODNs to treat various diseases.

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.

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.

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) 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.

Adjuvants are crucial components of vaccines. They significantly improve vaccine efficacy by modulating, enhancing, or extending the immune response and at the same time reducing the amount of antigen needed. In contrast to previously licensed adjuvants, current successful adjuvant formulations often consist of several molecules, that when combined, act synergistically by activating a variety of immune mechanisms. These “combination adjuvants” are already registered with several vaccines, both in humans and animals, and novel combination adjuvants are in the pipeline. With improved knowledge of the type of immune responses needed to successfully induce disease protection by vaccination, combination adjuvants are particularly suited to not only enhance, but also direct the immune responses desired to be either Th1-, Th2- or Th17-biased. Indeed, in view of the variety of disease and population targets for vaccine development, a panel of adjuvants will be needed to address different disease targets and populations. Here, we will review well-known and new combination adjuvants already licensed or currently in development—including ISCOMs, liposomes, Adjuvant Systems Montanides, and triple adjuvant combinations—and summarize their performance in preclinical and clinical trials. Several of these combination adjuvants are promising having promoted improved and balanced immune responses.

Bovine viral diarrhea virus (BVDV) is a pathogen of major importance in cattle, so there is a need for new effective vaccines. DNA vaccines induce balanced immune responses and are relatively inexpensive and thus promising for both human and veterinary applications. In this study, newborn calves with maternal antibodies were vaccinated intramuscularly (i.m.) with a BVDV E2 DNA vaccine with the TriGrid Delivery System for i.m. delivery (TDS-IM). Two doses of this vaccine spaced 6 or 12 weeks apart were sufficient to induce significant virus-neutralizing antibody titers, numbers of activated T cells, and reduction in viral shedding and clinical presentations after BVDV-2 challenge. In contrast to the placebo-treated animals, the vaccinated calves did not lose any weight, which is an excellent indicator of the well-being of an animal and has a significant economic impact. Furthermore, the interval between the two vaccinations did not influence the magnitude of the immune responses or degree of clinical protection, and a third immunization was not necessary or beneficial. Since electroporation may enhance not only the magnitude but also the duration of immunity after DNA immunization, the interval between vaccination and challenge was extended in a second trial, which showed that two doses of this E2 DNA vaccine again significantly reduced clinical disease against BVDV for several months. These results are promising and support this technology for use against infectious diseases in cattle and large species, including humans, in general.

Respiratory syncytial virus (RSV) is the leading cause of infant bronchiolitis. The closely related pneumonia virus of mice (PVM) causes a similar immune-mediated disease in mice, which allows an analysis of host factors that lead to severe illness. This project was designed to compare the immune responses to lethal and sublethal doses of PVM strain 15 in Balb/c and C57Bl/6 mice. Balb/c mice responded to PVM infection with an earlier and stronger innate response that failed to control viral replication. Production of inflammatory cyto- and chemokines, as well as infiltration of neutrophils and IFN-γ secreting natural killer cells into the lungs, was more predominant in Balb/c mice. In contrast, C57Bl/6 mice were capable of suppressing both viral replication and innate inflammatory responses. After a sublethal infection, PVM-induced IFN-γ production by splenocytes was stronger early during infection and weaker at late time points in C57Bl/6 mice when compared to Balb/c mice. Furthermore, although the IgG levels were similar and the mucosal IgA titres lower, the virus neutralizing antibody titres were higher in C57Bl/6 mice than in Balb/c mice. Overall, the difference in susceptibility of these two strains appeared to be related not to an inherent T helper bias, but to the capacity of the C57Bl/6 mice to control both viral replication and the immune response elicited by PVM.

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.

linked severe combined immunodeficiency (SCID-X1) is a profound deficiency of T, B, and natural killer (NK) cell immunity caused by mutations in IL2RG encoding the common chain (γc) of several interleukin receptors. Gamma-retroviral (γRV) gene therapy of SCID-X1 infants without conditioning restores T cell immunity without B or NK cell correction, but similar treatment fails in older SCID-X1 children. We used a lentiviral gene therapy approach to treat five SCID-X1 patients with persistent immune dysfunction despite haploidentical hematopoietic stem cell (HSC) transplant in infancy. Follow-up data from two older patients demonstrate that lentiviral vector γc transduced autologous HSC gene therapy after nonmyeloablative busulfan conditioning achieves selective expansion of gene-marked T, NK, and B cells, which is associated with sustained restoration of humoral responses to immunization and clinical improvement at 2 to 3 years after treatment. Similar gene marking levels have been achieved in three younger patients, albeit with only 6 to 9 months of follow-up. Lentiviral gene therapy with reduced-intensity conditioning appears safe and can restore humoral immune function to posthaploidentical transplant older patients with SCID-X1.

Adjuvants are critical components of many vaccines. The majority of existing vaccines contain a single adjuvant. Owing to their inherent limitations, no single adjuvant is capable of inducing all the protective immune responses required in the many different vaccines. Consequently, investigators are exploring the potential of using formulations with multiple adjuvants in a vaccine. An emerging paradigm is that careful selection of adjuvant combinations can result in complementary and even synergistic enhancement of immune responses to vaccines. This approach is promising and presents tremendous opportunities for vaccinologists to tailor immune responses to specific vaccines. In this article, adjuvant combinations at different stages of development will be reviewed.

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.

Summary. 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.

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.

Bovine respiratory syncytial virus (BRSV) infects cells of the respiratory mucosa, so it is desirable to develop a vaccination strategy that induces mucosal immunity. To achieve this, various delivery routes were compared for formalin-inactivated (FI) BRSV formulated with CpG oligodeoxynucleotide (ODN) and polyphosphazene (PP). Intranasal delivery of the FI-BRSV formulation was superior to subcutaneous delivery in terms of antibody, cell-mediated, and mucosal immune responses, as well as reduction in virus replication after BRSV challenge. Although intranasal delivery of FI-BRSV also induced higher serum and lung antibody titers and gamma interferon (IFN-γ) production in the lungs than intranasal-subcutaneous and/or subcutaneous-intranasal prime-boost strategies, no significant differences were observed in cell-mediated immune responses or virus replication in the lungs of challenged mice. Interleukin 5 (IL-5), eotaxin, and eosinophilia were enhanced after BRSV challenge in the lungs of subcutaneously immunized mice compared to unvaccinated mice, but not in the lungs of mice immunized intranasally or through combinations of the intranasal and subcutaneous routes. These results suggest that two intranasal immunizations with FI-BRSV formulated with CpG ODN and PP are effective and safe as an approach to induce systemic and mucosal responses, as well to reduce virus replication after BRSV challenge. Furthermore, intranasal-subcutaneous and subcutaneous-intranasal prime-boost strategies were also safe and almost as efficacious. In addition to the implications for the development of a protective BRSV vaccine for cattle, formulation with CpG ODN and PP could also prove important in the development of a mucosal vaccine that induces protective immunity against human RSV.

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.

CpG oligodeoxynucleotides (ODN) activate the immune system and are promising immunotherapeutic agents against infectious diseases, allergy/asthma and cancer. It has become apparent that while CpG ODN are potent immune activators in mice, their immune stimulatory effects are often less dramatic in humans and large animals. This disparity between rodents and mammals has been attributed to the differences in TLR9 expression in different species. This along with the sometimes transient activity of ODN may limit its potential immunotherapeutic applications. Several approaches to enhance the activity of CpG ODN have been explored including formulation of ODN in depot-forming adjuvants, and more recently, coadministration with polyphosphazenes, inhibitors of cytokines that downregulate TLR9 activation, and simultaneous activation with multiple TLR agonists. We will discuss these approaches and the mechanisms involved, with emphasis on what we have learned from large animal models.

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.

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-γ-secreting cells in the peripheral blood. Depletion studies showed that CD4+ T cells were responsible for IFN-γ 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.

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.

Bovine herpesvirus-1 (BHV-1) is one of the major respiratory pathogens in cattle worldwide. Although antibodies have been correlated with protection and recovery from BHV-1 infection, the cell-mediated immune response is also a critical defense mechanism because cell-to-cell spread occurs before hematogenous spread. Furthermore, induction of robust T-cell memory is critical for the long-term duration of immunity. Among current commercial vaccines, the attenuated conventional vaccines induce a balanced immune response and long-term memory but may result in viral shedding. By contrast, inactivated vaccines primarily elicit a humoral immune response and relative short-term memory. These vaccines do not allow differentiation of vaccinated from infected cattle. Recent efforts are focusing on the development of vaccines that induce a balanced immune response and long-term memory, as well as having differentiation markers. This includes well-defined genetically engineered gene-deleted, subunit and vectored vaccines.

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.

The pneumonia virus of mice (PVM) model is used to study respiratory syncytial virus (RSV) pathogenesis. The outcome of PVM infection varies in different inbred mouse strains, BALB/c being highly susceptible and C57BL/6 more resistant. As the disease symptoms induced by RSV infection can become more severe as people age, we examined the primary and secondary immune responses to infection with PVM in aged BALB/c and C57BL/6 mice. Based on clinical parameters, aged C57BL/6 mice displayed less severe disease than young adult mice when infected with 3000pfu of PVM-15, while BALB/c mice were equally susceptible at both ages showing significant weight loss and high levels of virus replication. Furthermore, after primary infection the CD4(+) T cell numbers in the lungs were higher in young adult mice, while the CD8(+) T cell numbers were comparable in both age groups and strains. When either C57BL/6 or BALB/c mice were infected with PVM as young adults and then re-infected as aged mice, they were protected from clinical disease, while virus replication was reduced. In contrast to mice with a primary PVM-infection, re-infected mice did not have infiltration of neutrophils or inflammatory mediators in the lung. BALB/c mice had higher virus neutralizing antibody levels in the serum and lung than C57BL/6 mice upon re-infection. Re-infection with PVM led to significant influx of effector CD4(+) T cells into the lungs when compared to aged mice with a primary infection, while this cell population was decreased in the lung draining lymph nodes in both mouse strains. After re-infection the effector CD8(+) T cell population was also decreased in the lung draining lymph nodes in both mouse strain when compared to aged mice after primary infection. However, the central memory CD4(+) and CD8(+) T cells were significantly enhanced in numbers in the lungs and draining lymph nodes of both mouse strains after re-infection, and these numbers were higher for C57BL/6 mice.

Vaccination of calves with formalin-inactivated bovine respiratory syncytial virus (FI-BRSV) induces low levels of cellular immunity that may not be protective. Since inactivated and subunit vaccines formulated with CpG oligodeoxynucleotides (ODNs) have been shown to induce cellular immune responses, we studied the ability of a FI-BRSV vaccine formulated with CpG ODN to elicit cellular immunity against BRSV. Neonatal calves were immunized with FI-BRSV, FI-BRSV formulated with CpG ODN or medium and challenged with BRSV after two immunizations. Calves vaccinated with FI-BRSV formulated with CpG ODN developed increased numbers of IFN-gamma secreting cells in the peripheral blood and broncho-tracheal lymph nodes and enhanced BRSV-specific serum IgG2 in comparison to FI-BRSV immunized animals. Calves that received the FI-BRSV vaccine formulated with CpG ODN also experienced a reduction in the amount of BRSV in the lung tissue. Based on these observations, CpG ODN appears to be a suitable candidate adjuvant for inactivated BRSV vaccines.

International audience; The viral envelope glycoprotein D from bovine herpesviruses 1 and 5 (BoHV-1 and -5), two important pathogens of cattle, is a major component of the virion and plays a critical role in the pathogenesis of herpesviruses. Glycoprotein D is essential for virus penetration into permissive cells and thus is a major target for virus neutralizing antibodies during infection. In view of its role in the induction of protective immunity, gD has been tested in new vaccine development strategies against both viruses. Subunit, DNA and vectored vaccine candidates have been developed using this glycoprotein as the primary antigen, demonstrating that gD has the capacity to induce robust virus neutralizing antibodies and strong cell-mediated immune responses, as well as protection from clinical symptoms, in target species. This review highlights the structural and functional characteristics of BoHV-1, BoHV-5 and where appropriate, Human herpesvirus gD, as well as its role in viral entry and interactions with host cell receptors. Furthermore, the interactions of gD with the host immune system are discussed. Finally, the application of this glycoprotein in new vaccine design is reviewed, taking its structural and functional characteristics into consideration.

Intercellular spread of bovine herpesvirus-1 (BHV-1) VP22 was demonstrated in living COS-7 cells transfected with a plasmid expressing VP22-YFP (yellow fluorescence protein) and CFP (cyan fluorescence protein) bicistronically. The intercellular trafficking property of VP22 was localized to the C-terminal portion of the molecule (amino acids 121-258; VP22-C). Plasmids encoding a truncated form of BHV-1 glycoprotein D (tgD) fused to VP22, VP22-C, or the N-terminal portion of VP22 (amino acids 1-120; VP22-N) were constructed. Mice immunized with plasmid encoding tgD-VP22 or tgD-VP22-C developed stronger immune responses when compared to animals immunized with plasmid encoding tgD or tgD fused to tgD-VP22-N.

Virus-specific cytotoxic T lymphocytes (CTLs) are considered to be important in protection against and recovery from viral infections. In this study, several approaches to induce cytotoxicity against bovine herpesvirus 1 (BHV-1) were evaluated. Vaccination of C57BL/6 mice with BHV-1 induced a strong humoral, but no CTL, response, which may be due to downregulation of major histocompatibility complex class I molecules. In contrast, vaccinia virus expressing glycoprotein B (gB) elicited a weaker antibody response, but strong cytotoxicity, in mice. As an approach to inducing both strong humoral and cellular immune responses, a plasmid vector was then used to express gB. Both antibody and CTL responses were induced by the plasmid encoding gB in C57BL/6 and C3H mice, regardless of the type of vector backbone. This demonstrated that DNA immunization induces a broad-based immune response to BHV-1 gB. Interestingly, removal of the membrane anchor, which resulted in secretion of gB from transfected cells, did not result in reduced cytotoxicity. Here, it is shown that, compared with the cell-associated counterpart, plasmid-encoded secreted protein may induce enhanced immune responses in cattle. Therefore, calves were immunized intradermally with pMASIAtgB, a plasmid encoding the secreted form of gB (tgB), using a needle-free injection system. This demonstrated that pMASIAtgB elicited both humoral responses and activated gamma interferon-secreting CD8+ CTLs, suggesting that a DNA vaccine expressing tgB induces a CTL response in the natural host of BHV-1.

Commercial killed bovine respiratory syncytial virus (K-BRSV) and formalin-inactivated BRSV (FI-BRSV) tend to induce Th2-type immune responses, which may not be protective and may even be detrimental during subsequent exposure to the virus. In this study we assessed the ability of CpG oligodeoxynucleotides (ODNs) to aid in the generation of effective and protective BRSV-specific immune responses. Mice were immunized subcutaneously with FI-BRSV formulated with CpG ODN, Emulsigen (Em), CpG ODN and Em, or non-CpG ODN and Em. Two additional groups were immunized with K-BRSV or K-BRSV and CpG ODN. After two vaccinations, the mice were challenged with BRSV. FI-BRSV induced Th2-biased immune responses characterized by production of serum immunoglobulin G1 (IgG1) and IgE, as well as interleukin-4 (IL-4), by in vitro-restimulated splenocytes. Formulation of FI-BRSV with CpG ODN, but not with non-CpG ODN, enhanced serum IgG2a and IFN-γ production by splenocytes, whereas serum IgE was reduced. Although the immune response induced by K-BRSV was not as strongly Th2 biased, the addition of CpG ODN to this commercial vaccine also resulted in a more Th1-type response. Furthermore, the addition of CpG ODN to the BRSV vaccine formulations resulted in enhanced neutralizing antibody responses. Significant production of IL-5, eotaxin, and eosinophilia was observed in the lungs of FI-BRSV- and K-BRSV-immunized mice. However, IL-5 and eotaxin levels, as well as the number of eosinophils, were decreased in the mice vaccinated with the CpG ODN-formulated vaccines. Finally, when formulated with CpG ODN, both FI-BRSV and K-BRSV significantly reduced virus production after challenge with BRSV.

For this study, the intercellular trafficking ability of bovine herpesvirus 1 (BHV-1) VP22 was applied to improve the efficacy of a DNA vaccine in calves. A plasmid encoding a truncated version of glycoprotein D (tgD) fused to VP22 was constructed. The plasmid encoding tgD-VP22 elicited significantly enhanced and more balanced immune responses than those induced by a plasmid encoding tgD. Furthermore, protection against a BHV-1 challenge was obtained in calves immunized with the plasmid encoding tgD-VP22, as shown by significant reductions in viral excretion. However, less significant protection was observed for animals vaccinated with the tgD-expressing plasmid, correlating with the lower level of immunity observed prechallenge. This is the first report of the use of VP22 as a transport molecule in the context of a DNA vaccine for a large animal species.

Plasmid DNA continues to attract interest as a potential vaccine-delivery vehicle. However, the mechanisms whereby immune responses are elicited by plasmids are not fully understood. Although there have been suggestions regarding the importance of CpG motifs in plasmid immunogenicity, the molecular mechanisms by which CpG motifs enhance immune responses to DNA vaccines are not well understood. As Toll-like receptor 9-deficient (TLR9-/-) mice fail to respond to the adjuvant effects of CpG oligonucleotides, we used these mice to determine the effect of CpG motifs in plasmids used for DNA immunization. In the study described below, we report that DNA immunization was as effective in eliciting antigen-specific antibody and at stimulating antigen-specific interferon-gamma (IFN-gamma)-secreting cells in TLR9-/- mice as in TLR9+/+ mice. This study illustrates that DNA vaccines elicit immune responses by multiple mechanisms and demonstrates that TLR9 is not essential for the induction of immune responses following DNA immunization.

Cell-mediated immune responses to hepatitis C virus (HCV) proteins play a key role in recovery from infection. The NS3 protein of HCV is of special interest, since it is one of the most conserved proteins and NS3-specific immune responses are stronger and more frequently observed in patients resolving the infection than in chronically infected patients. Since these characteristics make NS3 an attractive vaccine candidate, the objective of this study was to optimize NS3-specific immune responses. Results from this group first demonstrated that a plasmid enriched with 24 CpG motifs (pBISIA24-NS3) tends to induce the strongest and most consistent Th1-biased immune response. Subsequently, it was shown that NS3 formulated with CpG oligodeoxynucleotide and Quil A (rNS3+CpG+Quil A) adjuvants induces a balanced immune response in mice, whereas rNS3 combined with either CpG or Quil A elicits a Th2-biased response. To further enhance NS3-specific cell-mediated immune responses, a vaccination regime consisting of priming with pBISIA24-NS3, followed by boosting with rNS3+CpG+Quil A, was explored in mice and pigs. When compared to immunization with rNS3+CpG+Quil A, this regime shifted the immune response to a Th1-type response and, accordingly, enhanced MHC I-restricted killing by cytotoxic T lymphocytes in mice. Although immunization with pBISIA24-NS3 also induced a Th1-biased response, including cytotoxicity in the mice, the humoral response was significantly lower than that induced by the DNA prime–protein boost regime. These results demonstrate the advantage of a DNA prime–protein boost approach in inducing a strong NS3-specific cell-mediated, as well as humoral, immune response, in both inbred laboratory and outbred large animal species.

The objective of this study was to determine whether a DNA vaccine encoding bovine CD154 linked to a truncated version of bovine herpesvirus-1 (BHV-1) glycoprotein D (tgD-CD154) induces enhanced tgD-specific immune responses in cattle. In vitro characterization demonstrated that tgD and tgD-CD154 both bind to cultured bovine B cells, whereas only tgD-CD154 induces interleukin-4-dependent proliferation, suggesting that tgD-CD154 specifically binds the CD40 receptor and induces receptor signalling. Calves were immunized with plasmid encoding either tgD or tgD-CD154 by intradermal injection with a needle-free device. After two immunizations, tgD-specific immune responses were observed in both vaccinated groups and after challenge with BHV-1 these responses further increased. Animals immunized with plasmid encoding tgD-CD154 had significantly higher tgD-specific serum titres of immunoglobulins G and A but significantly lower numbers of tgD-specific interferon-gamma-secreting cells than animals immunized with plasmid encoding tgD after BHV-1 challenge. This suggests that the expression of an antigen as a chimeric protein with CD154 can qualitatively alter immune responses in cattle. Since we previously showed that plasmid encoding tgD-CD154 induces significantly enhanced secondary tgD-specific antibody responses in sheep, there appear to be interspecies differences in the immune responses induced by tgD-CD154, which suggests that both proteins in the chimeric molecule may influence protein targeting and the induction of an immune response.

DNA vaccines consist of antigen-encoding bacterial plasmids that are capable of inducing antigen-specific immune responses upon inoculation into a host. This method of immunization is advantageous in terms of simplicity, adaptability, and cost of vaccine production. However, the entry of DNA vaccines and expression of antigen are subjected to physical and biochemical barriers imposed by the host. In small animals such as mice, the host-imposed impediments have not prevented DNA vaccines from inducing long-lasting, protective humoral, and cellular immune responses. In contrast, these barriers appear to be more difficult to overcome in large animals and humans. The focus of this article is to summarize the limitations of DNA vaccines and to provide a comprehensive review on the different strategies developed to enhance the efficacy of DNA vaccines. Several of these strategies, such as altering codon bias of the encoded gene, changing the cellular localization of the expressed antigen, and optimizing delivery and formulation of the plasmid, have led to improvements in DNA vaccine efficacy in large animals. However, solutions for increasing the amount of plasmid that eventually enters the nucleus and is available for transcription of the transgene still need to be found. The overall conclusions from these studies suggest that, provided these critical improvements are made, DNA vaccines may find important clinical and practical applications in the field of vaccination.

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.

Bacterial DNA and synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs in particular sequence contexts (CpG ODN) are recognized as a danger signal by the innate immune system of vertebrates. For this reason, CpG ODNs have a potential application as both an adjuvant and nonspecific immune modulator and are currently being evaluated in a number of human and veterinary clinical trials. Given their potent immunostimulatory activity, CpG ODNs could possibly induce adverse reactions. As all adjuvants and immune modulators must be nontoxic to meet safety requirements, it was essential to address the safety aspects of CpG ODNs. The current review summarizes experiments carried out to date to establish the safety of CpG ODNs in animals.

The potential of CpG-enhanced plasmid DNA vectors encoding a truncated secreted form of bovine herpesvirus-1 (BHV-1) glycoprotein D (tgD) to induce enhanced immune responses in cattle was investigated. We created tgD expression plasmids containing 0, 40 or 88 copies of the hexamer 5′ GTCGTT 3′, a known pan-activating CpG motif in several species. The total tgD-specific IgG titre of calves immunized with these plasmids did not correlate with the CpG content of the plasmid backbone. However, the pBISIA88-tgD-vaccinated group showed a significantly lower IgG1:IgG2 ratio than calves immunized with pBISIA40-tgD or pMASIA-tgD, which has no CpG motifs inserted. Antigen-specific lymphocyte proliferation and IFN-γ secretion by peripheral blood mononuclear cells correlated positively with the CpG content of the vectors. In contrast, calves that received a killed BHV-1 vaccine had an IgG1-predominant isotype and low lymphocyte proliferation and IFN-γ levels. Following challenge, the pBISIA88-tgD-immunized group developed the greatest anamnestic response, the highest BHV-1 neutralization titres in serum and a significantly lower level of virus shedding than the saline control group. However, there were no significant differences in clinical symptoms of infection between the DNA-immunized groups and the saline control group. These data indicate that CpG-enhanced plasmids induce augmented immune responses and could be used to vaccinate against pathogens requiring a strong cellular response for protection.

The immunogenicity and protective efficacy of a bovine herpesvirus 1 (BHV-1) subunit vaccine formulated with Emulsigen (Em) and a synthetic oligodeoxynucleotide containing unmethylated CpG dinucleotides (CpG ODN) was determined in cattle. A truncated, secreted version of BHV-1 glycoprotein D (tgD) formulated with Em and CpG ODN at concentrations of 25, 2.5, or 0.25 mg/dose produced a more balanced immune response, higher levels of virus neutralizing antibodies, and greater protection after BHV-1 challenge compared to tgD adjuvanted with either Em or CpG ODN alone. In contrast, tgD formulated with Em and either 25 mg of a non-CpG ODN or another immunostimulatory compound, dimethyl dioctadecyl ammonium bromide, induced similar immunity and protection compared to tgD formulated with Em alone, a finding which confirms the immunostimulatory effect of ODN to be CpG motif mediated. Our results demonstrate the ability of CpG ODN to induce a strong and balanced immune response in a target species.

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.

Protective efficacy against bovine herpesvirus 1 (BoHV-1) has been demonstrated to be induced by a plasmid encoding bovine neutrophil beta-defensin 3 (BNBD3) as a fusion construct with truncated glycoprotein D (tgD). However, in spite of the increased cell-mediated immune responses induced by this DNA vaccine, the clinical responses of BoHV-1-challenged cattle were not reduced over those observed in animals vaccinated with the plasmid encoding tgD alone; this might have been because the vaccine failed to improve humoral responses. We hypothesized that an alternative vaccine design strategy that utilized the DNA vaccine pMASIA-tgD as a complex with BNBD3 might improve humoral responses while maintaining robust Th1-type cell-mediated responses. C57BL/6 mice were vaccinated with pMASIA-tgD complexed with 0, 0.01875, 0.1875, or 1.875 nmol of a stable synthesized analog of BNBD3 (aBNBD3). The best results were seen in mice immunized with the vaccine composed of pMASIA-tgD complexed to 0.1875 nmol aBNBD3. In this group, humoral responses were improved, as evidenced by increased virus neutralization, tgD-specific early IgG1, and later IgG2a titers, while the strong cell-mediated immune responses, measured based on specific gamma interferon (IFN-γ)-secreting cells, were maintained relative to pMASIA-tgD. Modulation of the immune response might have been due in part to the effect of BNBD3 on dendritic cells (DCs).In vitrostudies showed that murine bone marrow-derived DCs (BMDCs) pretreated with aBNBD3 were activated, as evidenced by CD11c downregulation, and were functionally mature, as shown by increased allostimulatory ability. Native, synthetic, and analog forms of BNBD3 were equally capable of inducing functional maturation of BMDCs.

Bovine herpesvirus 1 (BoHV-1) causes recurrent respiratory and genital infections in cattle and predisposes them to lethal secondary infections. While modified live and killed BoHV-1 vaccines exist, these are not without problems. Development of an effective DNA vaccine for BoHV-1 has the potential to address these issues. As a strategy to enhance DNA vaccine immunity, a plasmid encoding the bovine neutrophil beta-defensin 3 (BNBD3) as a fusion with truncated glycoprotein D (tgD) and a mix of two plasmids encoding BNBD3 and tgD were tested in mice and cattle. In mice, coadministration of BNBD3 on the separate plasmid enhanced the tgD-induced gamma interferon (IFN-γ) response but not the antibody response. BNBD3 fused to tgD did not affect the antibody levels or the number of IFN-γ-secreting cells but increased the induction of tgD-specific cytotoxic T lymphocytes (CTLs). In cattle, the addition of BNBD3 as a fusion construct also modified the immune response. While the IgG and virus-neutralizing antibody levels were not affected, the number of IFN-γ-secreting cells was increased after BoHV-1 challenge, specifically the CD8 + IFN-γ + T cells, including CD8 + IFN-γ + CD25 + CTLs. While reduced virus shedding, rectal temperature, and weight loss were observed, the level of protection was comparable to that observed in pMASIA-tgD-vaccinated animals. These data show that coadministration of BNBD3 with a protective antigen as a fusion in a DNA vaccine strengthened the Th1 bias and increased cell-mediated immune responses but did not enhance protection from BoHV-1 infection.

DNA vaccines have several advantages over conventional vaccines. One of the most important characteristics is the presentation of antigen via both MHC class I and class II receptors. Although this generally results in strong T-cell responses, antibody production and protection achieved by DNA immunization are unfortunately not always adequate. In contrast, modified live virus (MLV) vaccines usually induce adequate antibody and moderate cellular responses, whereas killed vaccines tend to elicit weak immune responses in general. A DNA prime–MLV boost regimen should result in enhanced cellular immunity and possibly improved antibody production. To test this hypothesis, plasmids encoding bovine herpesvirus-1 (BHV-1) glycoproteins B and D were delivered by gene gun to the genital mucosa of cattle prior to immunization with modified live BHV-1 vaccine. The immune responses induced were compared to those of an MLV-vaccinated group and a negative control group. Although significantly enhanced T-cell responses were induced by priming with the DNA vaccine, there was no increase in antibody titres. Similar levels of protection were induced by the MLV vaccine alone and the DNA prime and MLV boost regimen, which suggests that there is no correlation between the induction of T-cell responses and protection from BHV-1 challenge.

Oligodinucleotides containing CpG motifs stimulate vertebrate immune cells in vitro, have proven efficacy in murine disease models and are currently being tested in human clinical trials as therapies for cancer, allergy, and infectious disease. As there are no known immunostimulatory motifs for veterinary species, the potential of CpG DNA as a veterinary pharmaceutical has not been investigated. Here, optimal CpG motifs for seven veterinary and three laboratory species are described. The preferential recognition of a GTCGTT motif was strongly conserved across two vertebrate phyla, although a GACGTT motif was optimal for inbred strains of mice and rabbits. In a subsequent adjuvanticity trial, the in vitro screening methodology was validated in sheep, representing the first demonstration of CpG DNA efficacy in a veterinary species. These results should provide candidate immunostimulant and therapeutic drugs for veterinary use and enable the testing of CpG DNA in large animal models of human disease.

Mucosal surfaces are the primary sites for the transmission of infectious agents including viruses, so effective vaccines generally should induce mucosal immunity. Furthermore, noninvasive delivery is desirable because of the ease of application, the high degree of patient compliance, and the improved safety for patients and clinicians due to the elimination of needles. Unfortunately, most of the conventional vaccines are parenterally administered and result in systemic rather than mucosal immunity. Here we present the first report of mucosal immunity by noninvasive DNA immunization in a target species. As an approach to induce mucosal immunity against bovine herpesvirus-1, cows were immunized intravaginally with suppositories containing plasmid coding for glycoprotein D. Serum IgG, as well as IgA both in the serum and in the nasal fluids, were detected, which supports the contention of a common mucosal immune system. This level of immunity was of sufficient magnitude to minimize weight loss and significantly reduce the duration of virus shedding after intranasal viral challenge, which demonstrates the efficacy of suppository-based administration of DNA vaccines to target species. As this is a very practical method of delivery, it has great potential to be applied as vaccine or therapy in a variety of species.

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.

DNA-based vaccination constitutes one of the most recent approaches to vaccine development. This technology is in principle one of the most simple and yet versatile methods of inducing both humoral and cellular immune responses, as well as protection against a variety of infectious agents. However, although immune responses have been induced in a number of larger species, most information on the efficacy of DNA immunization has been generated in mice. In this review the information available to date about the use of DNA vaccines in farmed animals, including cattle, pigs and poultry, is presented. The areas that need specific attention in the future to bring this technology to the market are discussed, including the issues concerning delivery, safety, compatibility of plasmids in multivalent vaccines and the potential of using immune stimulants as part of a DNA vaccine.

Vaccination by a mucosal route is an excellent approach to the control of mucosally acquired infections. Several reports on rodents suggest that DNA vaccines can be used to achieve mucosal immunity when applied to mucosal tissues. However, with the exception of one study with pigs and another with horses, there is no information on mucosal DNA immunization of the natural host. In this study, the potential of inducing mucosal immunity in cattle by immunization with a DNA vaccine was demonstrated. Cattle were immunized with a plasmid encoding bovine herpesvirus 1 (BHV-1) glycoprotein B, which was delivered with a gene gun either intradermally or intravulvomucosally. Intravulvomucosal DNA immunization induced strong cellular immune responses and primed humoral immune responses. This was evident after BHV-1 challenge when high levels of both immunoglobulin G (IgG) and IgA were detected. Intradermal delivery resulted in lower levels of immunity than mucosal immunization. To determine whether the differences between the immune responses induced by intravulvomucosal and intradermal immunizations might be due to the efficacy of antigen presentation, the distributions of antigen and Langerhans cells in the skin and mucosa were compared. After intravulvomucosal delivery, antigen was expressed early and throughout the mucosa, but after intradermal administration, antigen expression occurred later and superficially in the skin. Furthermore, Langerhans cells were widely distributed in the mucosal epithelium but found primarily in the basal layers of the epidermis of the skin. Collectively, these observations may account for the stronger immune response induced by mucosal administration.