Local innate responses and protective immunity after intradermal immunization with bovine viral diarrhea virus E2 protein formulated with a combination adjuvant in cattle

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.     

Co-administration of polyphosphazenes with CpG oligodeoxynucleotides strongly enhances immune responses in mice immunized with Hepatitis B virus surface antigen

An emerging paradigm in vaccinology is that multiple adjuvant combinations may be more effective than individual adjuvants in enhancing immune responses to vaccine antigens. We investigated whether the polyphosphazenes used in combination with CpG oligodeoxynucleotides (ODN) were potent adjuvant formulations. BALB/c mice were immunized subcutaneously with Hepatitis B surface antigen (HBsAg) alone, or in various combinations with poly[di(sodium carboxylatophenoxy)phosphazene] (PCPP), poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) or CpG ODN. All three adjuvants enhanced HBsAg-specific IgG1 antibody responses with PCEP inducing the highest responses. PCEP and CpG ODN significantly enhanced the Th1-associated antibody isotype IgG2a. As expected CpG ODN induced predominantly Th1-type immune responses while PCEP was associated with mixed Th1/Th2 immune responses. Interestingly, PCEP and PCPP synergized with CpG ODN to further enhance antibody responses. Since the mechanisms which mediate the adjuvant activity of polyphosphazenes are not fully understood, we investigated whether PCEP and PCPP could stimulate innate immune responses. Incubation of mouse splenocytes with PCEP or PCPP (in the absence of antigen) stimulated production of IL-4 and IL-12, but only PCEP induced significant IFNγ production. Additionally, IL-12 was not required for PCEP induced IFNγ response. We conclude that the polyphosphazene–CpG ODN combination is a potent adjuvant formulation that is more effective in enhancing immune responses than either of the individual adjuvants. In addition, we provide evidence that PCEP and PCPP can stimulate innate cytokine production, suggesting a potential mechanism by which polyphosphazenes achieve their potent adjuvant effects.     

Homologous prime-boost with Zika virus envelope protein and poly (I:C) induces robust specific humoral and cellular immune responses

The recent outbreaks of Zika virus (ZIKV) infection and the potential association with Guillain-Barré syndrome in adults and with congenital abnormalities have highlighted the urgency for an effective vaccine. The ZIKV Envelope glycoprotein (E_ZIKV) is the most abundant protein on the virus surface, and has been evaluated together with the pre-membrane protein (prM) of the viral coat as a vaccine candidate in clinical trials. In this study, we performed a head-to-head comparison of the immune response induced by different E_ZIKV-based vaccine candidates in mice. We compared different platforms (DNA, recombinant protein), adjuvants (poly (I:C), CpG ODN 1826) and immunization strategies (homologous, heterologous).The hierarchy of adjuvant potency showed that poly (I:C) was a superior adjuvant than CpG ODN. While poly (I:C) assisted immunization reached a plateau in antibody titers after two doses, the CpG ODN group required an extra immunization dose. Besides, the administration of poly (I:C) induced higher E_ZIKV-specific cellular immune responses than CpG ODN. We also show that immunization with homologous prime-boost E_ZIKV protein + poly (I:C) regimen induced a more robust humoral response than homologous DNA (pVAX-E_ZIKV) or heterologous regimens (DNA/protein or protein/DNA). A detailed analysis of cellular immune responses revealed that homologous (E_ZIKV + poly (I:C)) and heterologous (pVAX-E_ZIKV/E_ZIKV + poly (I:C)) prime-boost regimens induced the highest magnitude of IFN-γ secreting cells and cytokine-producing CD4⁺ T cells.Overall, our data demonstrate that homologous E_ZIKV + poly (I:C) prime-boost immunization is sufficient to induce more robust specific-E_ZIKV humoral and cellular immune responses than the other strategies that contemplate homologous DNA (pVAX-E_ZIKV) or heterologous (pVAX-E_ZIKV/E_ZIKV + poly (I:C), and vice-versa) immunizations.     

Electroporation enhances immune responses and protection induced by a bovine viral diarrhea virus DNA vaccine in newborn calves with maternal antibodies

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.     

Comparative analysis of adaptive immune responses following experimental infections of cattle with bovine viral diarrhoea virus-1 and an Asiatic atypical ruminant pestivirus

Atypical ruminant pestiviruses are closely related to the two bovine viral diarrhoea virus (BVDV) species, BVDV-1 and BVDV-2. While there is evidence of cross-protective immune responses between BVDV-1 and BVDV-2, despite antigenic differences, there is little information on the antigenic cross-reactivity with atypical ruminant pestiviruses. The aim of this study was therefore to assess the specificity of antibody and T cell responses induced by experimental infection of calves with BVDV-1 strain Ho916, Th/04_KhonKaen (TKK), an Asiatic atypical ruminant pestivirus, or co-infection with both viruses. Homologous virus neutralization was observed in sera from both single virus infected and co-infected groups, while cross-neutralization was only observed in the TKK infected group. T cell IFN-γ responses to both viruses were observed in the TKK infected animals, whereas Ho916 infected calves responded better to homologous virus. Specifically, IFN-γ responses to viral non-structural protein, NS3, were observed in all infected groups while responses to viral glycoprotein, E2, were virus-specific. Broader antigen-specific cytokine responses were observed with similar trends between inoculation groups and virus species. The limited T cell and antibody immune reactivity of Ho916 inoculated animals to TKK suggests that animals vaccinated with current BVDV-1-based vaccines may not be protected against atypical ruminant pestiviruses.     

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

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

Immunization with PCEP microparticles containing pertussis toxoid, CpG ODN and a synthetic innate defense regulator peptide induces protective immunity against pertussis

We investigated the efficacy of a novel microparticle (MP) based vaccine formulation consisting of pertussis toxoid (PTd), polyphosphazene (PCEP), CpG ODN 10101 and synthetic cationic innate defense regulator peptide 1002 (IDR) against Bordetella pertussis in mice. We studied whether encapsulation of these IDR-CpG ODN complexes into polyphosphazene-based microparticles further enhanced their immunomodulatory activity compared to soluble formulations containing PCEP (SOL), or without PCEP (AQ). In vitro stimulation of murine macrophages showed MP induced significantly higher levels of pro-inflammatory cytokines. When assessed in a B. pertussis infection challenge model, a single immunization with MP formulation led to significantly lower bacterial loads compared to other formulations and non-vaccinated animals. ELISPOT of splenocytes showed that MP group mice had significantly higher number of antigen-specific IL-17 secreting cells. The cytokine profile in lung homogenates of MP group mice after challenge showed significantly higher amounts of MCP-1, TNF-α, IFN-γ, IL-12 and IL-17 and significantly lowered IL-10 levels suggesting a strong Th1 shift. Protection was observed against challenge infection with B. pertussis. On the other hand protective immune responses elicited in Quadracel^® immunized mice were Th2 skewed. Hence, we conclude that formulation of PTd, PCEP, CpG ODN and IDR into MP generates a protective immune response in mice against pertussis emphasizing the potential of MP as a delivery vehicle for the potential development of single-shot vaccines.     

Changes in circulating lymphocytes and lymphoid tissue associated with vaccination of colostrum deprived calves

The objective of this study was to compare immunological responses and lymphoid depletion in young, colostrum deprived calves following administration of vaccines containing modified-live bovine viral diarrhea virus (BVDV). A group of calves exposed to a typical virulence non-cytopathic (ncp) BVDV-2 field strain (ncp exposed) was included to compare responses of calves receiving vaccine to responses generated against a field strain (mimicking a natural infection). A negative control group administered a placebo was used in all comparisons. All vaccines used in the study were administered per manufacturer recommendations while ncp BVDV exposed calves received 5 ml intranasally (2.5 ml/nare; 4.2 × 10⁶ TCID₅₀/ml) of the BVDV-2 field strain. Samples collected at each time point included nasal swabs for virus detection, blood samples for complete blood counts and detection of viremia, PBMCs for flow cytometric analysis, serum for virus neutralization titers, and thymus tissue at necropsy for evaluation of lymphoid depletion. A measurable neutralizing BVDV titer was observed for all treatment groups excluding the control animals, which remained negative during the study period. Virus shedding was only detected from the ncp vaccinated and ncp exposed calves. A decline from baseline was observed for peripheral lymphocyte and CD4⁺ cells for the groups receiving the adjuvanted cytopathic (cp) vaccine, the double deleted genetically modified (ddGM) vaccine, the ncp vaccine and ncp exposed calves, but not for the control group or groups receiving cp vaccines. Thymus depletion was observed for the ncp vaccine and ncp exposed calves and to a lesser extent for the ddGM vaccine calves. Collectively, these data suggest that the virus biotype, method of attenuation, presentation, and use of adjuvant will influence vaccine impacts on lymphoid tissues and the immune response. As such, multiple variables should be considered when determining costs and benefits of vaccination.     

Vaccination with a modified-live bovine viral diarrhea virus (BVDV) type 1a vaccine completely protected calves against challenge with BVDV type 1b strains

Vaccination plays a significant role in the control of bovine viral diarrhea virus (BVDV) infection and spread. Recent studies revealed that type 1b is the predominant BVDV type 1 subgenotype, representing more than 75% of field isolates of BVDV-1. However, nearly all current, commercially available BVDV type 1 vaccines contain BVDV-1a strains. Previous studies have indicated that anti-BVDV sera, induced by BVDV-1a viruses, show less neutralization activity to BVDV-1b isolates than type 1a. Therefore, it is critically important to evaluate BVDV-1a vaccines in their ability to prevent BVDV-1b infection in calves. In current studies, calves were vaccinated subcutaneously, intradermally or intranasally with a single dose of a multivalent, modified-live viral vaccine containing a BVDV-1a strain, and were challenged with differing BVDV-1b strains to determine the efficacy and duration of immunity of the vaccine against these heterologous virus strains. Vaccinated calves, in all administration routes, were protected from respiratory disease caused by the BVDV-1b viruses, as indicated by significantly fewer clinical signs, lower rectal temperatures, reduced viral shedding and greater white blood cell counts than non-vaccinated control animals. The BVDV-1a vaccine elicited efficacious protection in calves against each BVDV-1b challenge strain, with a duration of immunity of at least 6 months.     

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

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

CpG DNA is an effective oral adjuvant to protein antigens in mice

We have previously reported that synthetic oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG ODN) are potent adjuvants to protein administered by intramuscular (IM) injection or intranasal (IN) inhalation to BALB/c mice. Herein, we have evaluated oral delivery of CpG ODN with purified hepatitis B surface antigen (HBsAg) or tetanus toxoid (TT) to determine its potential as an adjuvant to oral vaccines. CpG ODN augmented systemic (IgG in plasma, CTL, T-cell proliferation) and mucosal (IgA in lung, vaginal or gut washes, feces and saliva) immune responses against both antigens. CpG stimulated both T-helper type 1 (Th1) (CTL, IgG2a) and Th2 (IgG1, IgA) responses when delivered orally. Results from this study indicate that stimulatory CpG ODN may be effective as an adjuvant with oral vaccines.     

Induction of multispecific Th-1 type immune response against HCV in mice by protein immunization using CpG and Montanide ISA 720 as adjuvants

Recent studies demonstrate that Th1-type immune responses against a broad spectrum of hepatitis C virus (HCV) gene products are crucial to the resolution of acute HCV infection. We investigated new vaccine approaches to augment the strength of HCV-specific Th1-type immune responses. ELISPOT assay revealed that single or multiple protein immunization using both CpG ODN and Montanide ISA 720 as adjuvants induced much stronger IFN-gamma-producing Th1 responses against core, NS3 and NS5b targets than did the formulation without these adjuvants. Protein vaccination using CpG ODN and Montanide ISA 720 as adjuvants also greatly enhanced humoral responses to HCV core, E1/E2 and NS3. When specific IgG isotypes were assayed, protein immunization using CpG ODN and Montanide ISA 720 as adjuvants produced higher titers of IgG2a dominant antibodies than did protein immunization alone, indicating a more Th1-biased pathway. This increase in IgG2a is consistent with the induction of Th1 cells secreting IFN-gamma demonstrated by ELISPOT assay. In conclusion, protein immunization using CpG ODN and Montanide ISA 720 as adjuvants greatly enhanced cellular (Th1 type) as well as humoral immune responses against HCV in Balb/c mice. The use of adjuvants appears critical to the induction of Th1 immune responses during HCV vaccination with recombinant proteins.     

Poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) is a potent enhancer of mixed Th1/Th2 immune responses in mice immunized with influenza virus antigens

We investigated the ability of a novel polyphosphazene polyelectrolyte, poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) to enhance antigen-specific immune responses. BALB/c mice were immunized once subcutaneously with either bovine serum albumin (BSA) or influenza virus X:31 antigen alone, or in combination with PCEP, or either of the adjuvants poly[di(sodium carboxylatophenoxy)phosphazene] (PCPP) and alum. Both PCEP and PCPP significantly enhanced serum antigen-specific total IgG, IgG1 and IgG2a antibody titers, and these responses were highest in PCEP-immunized mice. Alum induced only a modest enhancement of antibody responses. Reducing the dose of X:31 antigen by 25-fold had no effect on antibody responses in mice immunized with PCPP and PCEP, but resulted in reduced titers in those immunized with alum. Analysis of X:31 antigen-specific cytokines revealed that alum and PCPP were associated with a predominantly IL-4 response. In contrast, PCEP was associated with production of both IFNgamma and IL-4. We conclude that PCEP is a potent enhancer of antigen-specific Th1 and Th2 immune responses and is a promising adjuvant for vaccine applications.     

DNA vaccination against bovine viral diarrhoea virus induces humoral and cellular responses in cattle with evidence for protection against viral challenge

The immune response induced by a DNA construct expressing the E2 envelope glycoprotein of bovine viral diarrhoea virus (BVDV) was studied in cattle. Four groups of five calves, were immunised by intradermal injection with a total of 1mg of plasmid DNA on each of two occasions, with a 3-week dose interval. Group 1 received non-coding plasmid DNA only (control), group 2 received the E2 coding plasmid (0.5mg) plus non-coding plasmid DNA (0.5mg) and groups 3 and 4 received the E2 coding plasmid plus plasmid encoding either bovine interleukin 2 (IL-2) or granulocyte macrophage colony stimulating factor (GM-CSF) respectively. Two weeks after the final immunisation, all calves were challenged by intranasal inoculation with 5 x 10(6) TCID(50) of homologous virus. On the day of challenge, neutralising antibodies were detectable in 13 of 15 vaccinated calves (one animal in each of groups 3 and 4 remained seronegative at this point). Thereafter, a strong anamnestic serological response was evident in all vaccinated animals. Furthermore, T-cell proliferation following in vitro re-stimulation with BVDV antigen was significantly elevated in the cytokine adjuvanted groups. This enhancement of BVDV specific immune responses in vaccinated animals was reflected in the clinical responses observed post-challenge. In particular, reduced febrile responses provided evidence of a disease sparing effect of vaccination. Significantly, whilst a transient viraemia was detected in all control animals following challenge, no virus was isolated from the leucocytes from 8 out of the 15 vaccinated animals. In groups 2 and 4, three animals remained virus free, although virus was isolated from two animals in each group at a single time point, while in group 3, three out of five animals had detectable viraemia.In summary, the administration of a DNA vaccine encoding only the E2 glycoprotein of BVDV induced a disease sparing effect in vaccinated calves following challenge and protected more than half of the vaccinated animals from detectable viraemia.     

One year duration of immunity of the modified live bovine viral diarrhea virus type 1 and type 2 and bovine herpesvirus-1 fractions of Vista® Once SQ vaccine

Three studies were performed to determine the duration of immunity of the bovine viral diarrhea virus type 1 and type 2 (BVDV-1 and BVDV-2) and bovine herpesvirus-1 (BHV-1) fractions of a commercially prepared modified-live vaccine. Vista^® Once SQ (Vista^®) vaccine contains five modified-live viruses, BVDV-1, BVDV-2, BHV-1, bovine respiratory syncytial virus, and bovine parainfluenza 3 virus, and two modified-live bacteria, Pasteurella multocida and Mannheimia haemolytica. For all three studies, calves were administered a single dose of vaccine or placebo vaccine subcutaneously, and were challenged with one of the three virulent viruses at least one year following vaccination. Calves were evaluated daily following challenge for clinical signs of disease associated with viral infection, nasal swab samples were evaluated for virus shedding, and serum was tested for neutralizing antibodies. Following the BVDV-1 and BVDV-2 challenges, whole blood was evaluated for white blood cell counts, and for the BVDV-2 study, whole blood was also evaluated for platelet counts. Calves vaccinated with BVDV type 1a, were protected from challenge with BVDV type 1b, and had significant reductions in clinical disease, fever, leukopenia, and virus shedding compared to control calves. Vaccinated calves in the BVDV-2 study were protected from clinical disease, mortality, fever, leukopenia, thrombocytopenia, and virus shedding compared to controls. Vaccinated calves in the BHV-1 study were protected from clinical disease and fever, and had significantly reduced duration of nasal virus shedding. These three studies demonstrated that a single administration of the Vista^® vaccine to healthy calves induces protective immunity against BVDV-1, BVDV-2 and BHV-1 that lasts at least one year following vaccination.     

Hepatitis C virus soluble E2 in combination with QuilA and CpG ODN induces neutralizing antibodies in mice

Several studies have emphasized the importance of an early, highly neutralizing antibody response in the clearance of Hepatitis C virus (HCV) infection. The envelope glycoprotein E2 is a major target for HCV neutralizing antibodies. Here, we compared antibody responses in mice immunized with native soluble E2 (sE2) from the H77 1a isolate coupled with different adjuvants or combinations of adjuvants. Adjuvanting sE2 with Freund's, monophosphoryl lipid A (MPL), cytosine phosphorothioate guanine oligodeoxynucleotide (CpG ODN), or alpha-galactosylceramide (αGalCer) derivatives elicited only moderate antibody responses. In contrast, immunizations with sE2 and QuilA elicited exceptionally high anti-E2 antibody titers. Sera from these mice effectively neutralized HCV pseudoparticles (HCVpp) 1a entry. Moreover, the combination of QuilA and CpG ODN further enhanced neutralizing antibody titers wherein cross-neutralization of HCVpp 4 was observed. We conclude that the combination of QuilA and CpG ODN is a promising adjuvant combination that should be further explored for the development of an HCV subunit vaccine. Our work also emphasizes that the ideal combination of adjuvant and immunogen has to be determined empirically.     

The STING activator c-di-AMP exerts superior adjuvant properties than the formulation poly(I:C)/CpG after subcutaneous vaccination with soluble protein antigen or DEC-205-mediated antigen targeting to dendritic cells

Vaccination is the most efficient strategy to protect from infectious diseases and the induction of a protective immune response not only depends on the nature of the antigen, but is also influenced by the vaccination strategy and the co-administration of adjuvants. Therefore, the precise monitoring of adjuvant candidates and their immune modulatory properties is a crucial step in vaccine development. Here, one central aspect is the induction of appropriate humoral and cellular effector mechanisms.In our study we performed a direct comparison of two promising candidates in adjuvant development, the STING activator bis-(3,5)-cyclic dimeric adenosine monophosphate (c-di-AMP) and the Toll-like receptor ligand formulation poly(I:C)/CpG. These were evaluated in C57BL/6 mice using the model antigen ovalbumin (OVA) in subcutaneous vaccination with soluble protein as well as in a dendritic cell (DC) targeting approach (αDEC-OVA). Strikingly, c-di-AMP as compared to poly(I:C)/CpG resulted in significantly higher antigen-specific IgG antibody levels when used in immunization with soluble OVA as well as in antigen targeting to DC. In vaccination with soluble OVA, c-di-AMP induced a significantly stronger CTL, Th1 and IFNγ-producing CD8⁺ memory T cell response than poly(I:C)/CpG. The response was CTL and Th1 cell dominated, a profile shared by both adjuvants. In the context of targeting OVA to DC, c-di-AMP induced significantly increased Th1 and Th2 cell responses as compared to poly(I:C)/CpG. Interestingly, the Th1 response dominated the overall T cell response only when c-di-AMP was used, indicating a distinct modulatory property of c-di-AMP when the DC targeting immunization approach was exploited.Taken together, we describe superior properties of c-di-AMP as compared to poly(I:C)/CpG in subcutaneous vaccination with soluble antigen as well as antigen targeting to DC. This indicates exceptionally effective adjuvant properties for c-di-AMP and provides compelling evidence of its potential for further adjuvant development, especially also when using DC targeting approaches.     

Development of an APC-targeted multivalent E2-based vaccine against Bovine Viral Diarrhea Virus types 1 and 2

The aim of this study was to develop and test a multivalent subunit vaccine against Bovine Viral Diarrhea Virus (BVDV) based on the E2 virus glycoprotein belonging to genotypes 1a, 1b and 2a, immunopotentiated by targeting these antigens to antigen-presenting cells. The E2 antigens were expressed in insect cells by a baculovirus vector as fusion proteins with a single chain antibody, named APCH I, which recognizes the β-chain of the MHC Class II antigen. The three chimeric proteins were evaluated for their immunogenicity in a guinea pig model as well as in colostrum-deprived calves. Once the immune response in experimentally vaccinated calves was evaluated, immunized animals were challenged with type 1b or type 2b BVDV in order to study the protection conferred by the experimental vaccine.The recombinant APCH I-tE21a-1b-2a vaccine was immunogenic both in guinea pigs and calves, inducing neutralizing antibodies. After BVDV type 1b and type 2 challenge of vaccinated calves in a proof of concept, the type 1b virus could not be isolated in any animal; meanwhile it was detected in all challenged non-vaccinated control animals. However, the type 2 BVDV was isolated to a lesser extent compared to unvaccinated animals challenged with type 2 BVDV. Clinical signs associated to BVDV, hyperthermia and leukopenia were reduced with respect to controls in all vaccinated calves. Given these results, this multivalent vaccine holds promise for a safe and effective tool to control BVDV in herds.     

Bovine viral diarrhea virus types 1 and 2 antibody response in calves receiving modified live virus or inactivated vaccines

Serums from calves receiving eight different commercial vaccines containing modified live virus (MLV) or inactivated bovine viral diarrhea virus (BVDV) immunogens were assayed for antibodies to types 1 and 2 BVDV strains. The immune response to the types 1 and 2 BVDV strains were evaluated in 48 calves receiving one of the eight vaccines for each group. For 7/8 vaccines, the BVDV vaccine immunogen was only type 1 whereas the remaining vaccine contained both types 1 and 2 immunogens. Calves administered MLV vaccine received only one dose at day 0, whereas those calves receiving the inactivated vaccines were administered two doses initially at days 0 and 28. Selected calves were revaccinated with only one dose at day 140. Animals vaccinated with type 1 vaccines developed titers to a broad range of type 1 BVDV, both cytopathic (CP) and noncytopathic (NCP) biotypes, with lower titers evident to type 2 BVDV strains. For some animals, the BVDV serum antibodies did not persist. Revaccination at day 140 induced a significant four-fold increase in animals with intermediate to low antibody titers. There did not appear to be any clear differences in antibody responses between type 1 MLV or inactivated vaccines. The calves receiving the inactivated vaccine containing types 1 and 2 had similar antibody levels to both types.