Novel Potent IFN-γ-Inducing CD8+ T Cell Epitopes Conserved among Diverse Bovine Viral Diarrhea Virus Strains.

Studies of immune responses elicited by bovine viral diarrhea virus (BVDV) vaccines have primarily focused on the characterization of neutralizing B cell and CD4+ T cell epitopes. Despite the availability of commercial vaccines for decades, BVDV prevalence in cattle has remained largely unaffected. There is limited knowledge regarding the role of BVDV-specific CD8+ T cells in immune protection, and indirect evidence suggests that they play a crucial role during BVDV infection. In this study, the presence of BVDV-specific CD8+ T cells that are highly cross-reactive in cattle was demonstrated. Most importantly, novel potent IFN-γ-inducing CD8+ T cell epitopes were identified from different regions of BVDV polyprotein. Eight CD8+ T cell epitopes were identified from the following structural BVDV Ags: Erns, E1, and E2 glycoproteins. In addition, from nonstructural BVDV Ags Npro, NS2-3, NS4A-B, and NS5A-B, 20 CD8+ T cell epitopes were identified. The majority of these IFN-γ-inducing CD8+ T cell epitopes were found to be highly conserved among more than 200 strains from BVDV-1 and -2 genotypes. These conserved epitopes were also validated as cross-reactive because they induced high recall IFN-γ+CD8+ T cell responses ex vivo in purified bovine CD8+ T cells isolated from BVDV-1- and -2-immunized cattle. Altogether, 28 bovine MHC class I-binding epitopes were identified from key BVDV Ags that can elicit broadly reactive CD8+ T cells against diverse BVDV strains. The data presented in this study will lay the groundwork for the development of a contemporary CD8+ T cell-based BVDV vaccine capable of addressing BVDV heterogeneity more effectively than current vaccines.

Opsonization but not pretreatment of equine macrophages with hyperimmune plasma nonspecifically enhances phagocytosis and intracellular killing of Rhodococcus equi.

BACKGROUND: Evidence regarding the efficacy of equine hyperimmune plasma to prevent pneumonia in foals caused by Rhodococcus equi is limited and conflicting. HYPOTHESIS: Opsonization with R. equi-specific hyperimmune plasma (HIP) will significantly increase phagocytosis and decrease intracellular replication of R. equi by alveolar macrophages (AMs) compared to normal plasma (NP). ANIMALS: Fifteen adult Quarter Horses were used to collect bronchoalveolar lavage cells. METHODS: In the first experiment, AMs from 9 horses were pretreated (incubated) with either HIP, NP, or media only (control) and then infected with nonopsonized R. equi. In a second experiment, AMs from 6 horses were infected with R. equi either opsonized with HIP or opsonized with NP. For both experiments, AMs were lysed at 0 and 48 hours and the number of viable R. equi quantified by culture were compared among groups using linear mixed-effects modeling with significance set at P < .05. RESULTS: Opsonization with either HIP or NP increased phagocytosis by AMs (P < .0001) and decreased intracellular survival of organisms in AMs (P < .0001). Pretreating AMs with either HIP or NP without opsonizing R. equi had no effects on phagocytosis or intracellular replication. CONCLUSIONS AND CLINICAL IMPORTANCE: Opsonizing R. equi with either NP or HIP decreases intracellular survival of organisms in AMs, but the effect does not appear to be enhanced by using HIP. Mechanisms other than effects on AMs must explain any clinical benefits of using HIP over NP to decrease the incidence of R. equi pneumonia in foals.

Mosaic Bovine Viral Diarrhea Virus Antigens Elicit Cross-Protective Immunity in Calves.

Bovine Viral Diarrhea Virus (BVDV) is an important pathogen that plays a significant role in initiating Bovine Respiratory Disease Complex (BRDC) in cattle. The disease causes multi-billion dollar losses globally due to high calf mortality and increased morbidity leading to heavy use of antibiotics. Current commercial vaccines provide limited cross-protection with several drawbacks such as safety, immunosuppression, potential reversion to virulence, and induction of neonatal pancytopenia. This study evaluates two prototype vaccines containing multiple rationally designed recombinant mosaic BVDV antigens for their potential to confer cross-protection against diverse BVDV strains. Genes encoding three novel mosaic antigens, designated E2123, NS2-31, and NS2-32, were designed in silico and expressed in mammalian cells for the formulation of a prototype protein-based vaccine. The mosaic antigens contain highly conserved protective epitopes from BVDV-1a, -1b, and -2, and included unique neutralizing epitopes from disparate strains to broaden coverage. We tested immunogenicity and protective efficacy of Expi293TM-expressed mosaic antigens (293F-E2123, 293F-NS2-31, and 293F-NS2-32), and baculovirus-expressed E2123 (Bac-E2123) mosaic antigen in calves. The Expi293TM-expressed antigen cocktail induced robust BVDV-specific cross-reactive IFN-γ responses, broadly neutralizing antibodies, and following challenge with a BVDV-1b strain, the calves had significantly (p < 0.05) reduced viremia and clinical BVD disease compared to the calves vaccinated with a commercial killed vaccine. The Bac-E2123 antigen was not as effective as the Expi293TM-expressed antigen cocktail, but it protected calves from BVD disease better than the commercial killed vaccine. The findings support feasibility for development of a broadly protective subunit BVDV vaccine for safe and effective management of BRD.

Adenovirus-vectored African Swine Fever Virus Antigens Cocktail Is Not Protective against Virulent Arm07 Isolate in Eurasian Wild Boar.

: African swine fever (ASF) is a viral disease of domestic and wild suids for which there is currently no vaccine or treatment available. The recent spread of ASF virus (ASFV) through Europe and Asia is causing enormous economic and animal losses. Unfortunately, the measures taken so far are insufficient and an effective vaccine against ASFV needs to be urgently developed. We hypothesized that immunization with a cocktail of thirty-five rationally selected antigens would improve the protective efficacy of subunit vaccine prototypes given that the combination of fewer immunogenic antigens (between 2 and 22) has failed to elicit protective efficacy. To this end, immunogenicity and efficacy of thirty-five adenovirus-vectored ASFV antigens were evaluated in wild boar. The treated animals were divided into different groups to test the use of BioMize adjuvant and different inoculation strategies. Forty-eight days after priming, the nine treated and two control wild boar were challenged with the virulent ASFV Arm07 isolate. All animals showed clinical signs and pathological findings consistent with ASF. This lack of protection is in line with other studies with subunit vaccine prototypes, demonstrating that there is still much room for improvement to obtain an effective subunit ASFV vaccine.

Adenovirus-vectored African Swine Fever Virus antigen cocktails are immunogenic but not protective against intranasal challenge with Georgia 2007/1 isolate.

African Swine Fever Virus (ASFV) causes a hemorrhagic disease in swine and wild boars with a fatality rate close to 100%. Less virulent strains cause subchronic or chronic forms of the disease. The virus is endemic in sub-Saharan Africa and an outbreak in Georgia in 2007 spread to Armenia, Russia, Ukraine, Belarus, Poland, Lithuania, and Latvia. In August 2018, there was an outbreak in China and in April 2019, ASFV was reported in Vietnam and Cambodia. Since no vaccine or treatment exists, a vaccine is needed to safeguard the swine industry. Previously, we evaluated immunogenicity of two adenovirus-vectored cocktails containing ASFV antigens and demonstrated induction of unprecedented robust antibody and T cell responses, including cytotoxic T lymphocytes. In the present study, we evaluated protective efficacy of both cocktails by intranasal challenge of pigs with ASFV-Georgia 2007/1. A nine antigen cocktail-(I) formulated in BioMize adjuvant induced strong IgG responses, but when challenged, the vaccinees had more severe reaction relative to the controls. A seven antigen cocktail-(II) was evaluated using two adjuvants: BioMize and ZTS-01. The BioMize formulation induced stronger antibody responses, but 8/10 vaccinees and 4/5 controls succumbed to the disease or reached experimental endpoint at 17 days post-challenge. In contrast, the ZTS-01 formulation induced weaker antibody responses, but 4/9 pigs succumbed to the disease while the 5 survivors exhibited low clinical scores and no viremia at 17 days post-challenge, whereas 4/5 controls succumbed to the disease or reached experimental endpoint. Overall, none of the immunogens conferred statistically significant protection.

Immunogenicity of a recombinant adenovirus expressing porcine reproductive and respiratory syndrome virus polyepitopes.

The objective of this work was to evaluate the immunogenicity of a chimeric antigen containing characterized PRRSV epitopes. A synthetic gene, designated HEJ, encoding defined epitopes was used to generate a recombinant adenovirus designed Ad-HEJ. The chimeric antigen included T-cell epitopes from structural and nonstructural proteins, and a neutralizing B-cell epitope. Following a homologous prime-boost immunization, the Ad-HEJ virus elicited significant (p<0.05) epitope-specific IFN-γ responses compared to sham-treatment. Two weeks post-challenge, this response was significantly (p<0.05) higher compared to the negative control treatment. IFN-γ response to PRRSV stimulation in vitro were observed in both groups only after challenge. Antibodies against PRRSV and peptides were detectable following prime-boost immunization in the Ad-HEJ treatment group and the responses increased post-challenge against the virus and against most of the peptides. All the swine were viremic one week post-challenge, but four weeks later, five out of the seven Ad-HEJ vaccinees had cleared the PRRSV, whereas only two of the six negative controls had cleared the virus. The outcome suggests that the adenovirus expressing defined epitopes induced a strong immune response against the peptides, but this response was not sufficient to confer protection against PRRSV challenge.

Adenovirus-vectored novel African Swine Fever Virus antigens elicit robust immune responses in swine.

African Swine Fever Virus (ASFV) is a high-consequence transboundary animal pathogen that often causes hemorrhagic disease in swine with a case fatality rate close to 100%. Lack of treatment or vaccine for the disease makes it imperative that safe and efficacious vaccines are developed to safeguard the swine industry. In this study, we evaluated the immunogenicity of seven adenovirus-vectored novel ASFV antigens, namely A151R, B119L, B602L, EP402RΔPRR, B438L, K205R and A104R. Immunization of commercial swine with a cocktail of the recombinant adenoviruses formulated in adjuvant primed strong ASFV antigen-specific IgG responses that underwent rapid recall upon boost. Notably, most vaccinees mounted robust IgG responses against all the antigens in the cocktail. Most importantly and relevant to vaccine development, the induced antibodies recognized viral proteins from Georgia 2007/1 ASFV-infected cells by IFA and by western blot analysis. The recombinant adenovirus cocktail also induced ASFV-specific IFN-γ-secreting cells that were recalled upon boosting. Evaluation of local and systemic effects of the recombinant adenovirus cocktail post-priming and post-boosting in the immunized animals showed that the immunogen was well tolerated and no serious negative effects were observed. Taken together, these outcomes showed that the adenovirus-vectored novel ASFV antigen cocktail was capable of safely inducing strong antibody and IFN-γ+ cell responses in commercial swine. The data will be used for selection of antigens for inclusion in a multi-antigen prototype vaccine to be evaluated for protective efficacy.

Characterization of a Broadly Reactive Anti-CD40 Agonistic Monoclonal Antibody for Potential Use as an Adjuvant.

Lack of safe and effective adjuvants is a major hindrance to the development of efficacious vaccines. Signaling via CD40 pathway leads to enhanced antigen processing and presentation, nitric oxide expression, pro-inflammatory cytokine expression by antigen presenting cells, and stimulation of B-cells to undergo somatic hypermutation, immunoglobulin class switching, and proliferation. Agonistic anti-CD40 antibodies have shown promising adjuvant qualities in human and mouse vaccine studies. An anti-CD40 monoclonal antibody (mAb), designated 2E4E4, was identified and shown to have strong agonistic effects on primary cells from multiple livestock species. The mAb recognize swine, bovine, caprine, and ovine CD40, and evoked 25-fold or greater proliferation of peripheral blood mononuclear cells (PBMCs) from these species relative to cells incubated with an isotype control (p<0.001). In addition, the mAb induced significant nitric oxide (p<0.0001) release by bovine macrophages. Furthermore, the mAb upregulated the expression of MHC-II by PBMCs, and stimulated significant (p<0.0001) IL-1α, IL6, IL-8, and TNF-α expression by PBMCs. These results suggest that the mAb 2E4E4 can target and stimulate cells from multiple livestock species and thus, it is a potential candidate for adjuvant development. This is the first study to report an anti-swine CD40 agonistic mAb that is also broadly reactive against multiple species.

Priming Cross-Protective Bovine Viral Diarrhea Virus-Specific Immunity Using Live-Vectored Mosaic Antigens.

Bovine viral diarrhea virus (BVDV) plays a key role in bovine respiratory disease complex, which can lead to pneumonia, diarrhea and death of calves. Current vaccines are not very effective due, in part, to immunosuppressive traits and failure to induce broad protection. There are diverse BVDV strains and thus, current vaccines contain representative genotype 1 and 2 viruses (BVDV-1 & 2) to broaden coverage. BVDV modified live virus (MLV) vaccines are superior to killed virus vaccines, but they are susceptible to neutralization and complement-mediated destruction triggered by passively acquired antibodies, thus limiting their efficacy. We generated three novel mosaic polypeptide chimeras, designated NproE2123; NS231; and NS232, which incorporate protective determinants that are highly conserved among BVDV-1a, 1b, and BVDV-2 genotypes. In addition, strain-specific protective antigens from disparate BVDV strains were included to broaden coverage. We confirmed that adenovirus constructs expressing these antigens were strongly recognized by monoclonal antibodies, polyclonal sera, and IFN-γ-secreting T cells generated against diverse BVDV strains. In a proof-of-concept efficacy study, the multi-antigen proto-type vaccine induced higher, but not significantly different, IFN-γ spot forming cells and T-cell proliferation compared to a commercial MLV vaccine. In regards to the humoral response, the prototype vaccine induced higher BVDV-1 specific neutralizing antibody titers, whereas the MLV vaccine induced higher BVDV-2 specific neutralizing antibody titers. Following BVDV type 2a (1373) challenge, calves immunized with the proto-type or the MLV vaccine had lower clinical scores compared to naïve controls. These results support the hypothesis that a broadly protective subunit vaccine can be generated using mosaic polypeptides that incorporate rationally selected and validated protective determinants from diverse BVDV strains. Furthermore, regarding biosafety of using a live vector in cattle, we showed that recombinant human adenovirus-5 was cleared within one week following intradermal inoculation.

Induction of Robust Immune Responses in Swine by Using a Cocktail of Adenovirus-Vectored African Swine Fever Virus Antigens.

The African swine fever virus (ASFV) causes a fatal hemorrhagic disease in domestic swine, and at present no treatment or vaccine is available. Natural and gene-deleted, live attenuated strains protect against closely related virulent strains; however, they are yet to be deployed and evaluated in the field to rule out chronic persistence and a potential for reversion to virulence. Previous studies suggest that antibodies play a role in protection, but induction of cytotoxic T lymphocytes (CTLs) could be the key to complete protection. Hence, generation of an efficacious subunit vaccine depends on identification of CTL targets along with a suitable delivery method that will elicit effector CTLs capable of eliminating ASFV-infected host cells and confer long-term protection. To this end, we evaluated the safety and immunogenicity of an adenovirus-vectored ASFV (Ad-ASFV) multiantigen cocktail formulated in two different adjuvants and at two immunizing doses in swine. Immunization with the cocktail rapidly induced unprecedented ASFV antigen-specific antibody and cellular immune responses against all of the antigens. The robust antibody responses underwent rapid isotype switching within 1 week postpriming, steadily increased over a 2-month period, and underwent rapid recall upon boost. Importantly, the primed antibodies strongly recognized the parental ASFV (Georgia 2007/1) by indirect fluorescence antibody (IFA) assay and Western blotting. Significant antigen-specific gamma interferon-positive (IFN-γ+) responses were detected postpriming and postboosting. Furthermore, this study is the first to demonstrate induction of ASFV antigen-specific CTL responses in commercial swine using Ad-ASFV multiantigens. The relevance of the induced immune responses in regard to protection needs to be evaluated in a challenge study.

Development and characterization of a monoclonal antibody against porcine CD205.

The aim of this work was to develop mAbs against porcine CD205 and to conduct a comparative analysis of the CD205 protein expression on lymphoid tissues, monocyte-derived dendritic cells (DCs) and DCs isolated from the porcine skin. To conduct this study, we generated a monoclonal antibody, designated 1.F6F6, against the C-type lectin-like domain-5 of the porcine CD205 and showed that it recognizes a protein band of ∼200 kDa by Western Blot analysis in mesenteric lymph nodes cells. Flow cytometric analysis showed that the mAb 1.F6F6 recognized 28.5%, 28.1% and 34.1% of cells from tonsil, inguinal and mesenteric lymph nodes, respectively, and 6% of cells from thymus. Analysis of monocyte-derived DCs showed that approximately 20% were positive and activation of the cells with LPS increased the positive population to 36%. Analysis of DCs isolated from the porcine skin showed that approximately 70% of the cell population expressed the CD205 receptor. The development of a monoclonal antibody capable of recognizing the CD205 receptor in swine opens up possibilities of applying new strategies for enhancing vaccine efficacy by using the anti-CD205 antibody for DC antigen-targeting to enhance priming of immune responses.

CD205 antigen targeting combined with dendritic cell recruitment factors and antigen-linked CD40L activation primes and expands significant antigen-specific antibody and CD4(+) T cell responses following DNA vaccination of outbred animals.

Dendritic cell antigen targeting primes robust immune responses in mouse models. Optimizing this immunization strategy in the actual hosts that require protection will advance development of efficacious contemporary vaccines. In a proof-of-concept study, we tested the immunogenicity of a single, low dose of a novel multi-component DNA construct expressing a CD205-targeted antigen fused to a CD40L minimal functional domain for linked DC activation. The DNA construct was formulated with DNA-encoded Flt3L and GM-CSF for DC recruitment and the formulation was evaluated in MHC class II-matched calves. Immunization of the calves with the CD205 antigen-targeting construct mixed with the cytokine constructs induced significant IFN-γ-secreting CD4(+) T-cells, CD4(+) T-cell proliferation, and antibody responses detectable within one week post-immunization. CD205 antigen-targeting significantly expanded IFN-γ-secreting CD4(+) T-cells, CD4(+) T-cell proliferation, and IgG antibody responses three weeks post-immunization. Nineteen weeks post-priming, the IFN-γ-secreting CD4(+) T-cells, CD4(+) T-cell proliferation, and the IgG titers were waning, but they remained significant. Following boosting at nineteen weeks post-immunization, the immune responses primed by the CD205-targeted antigen underwent rapid recall and the mean response tripled within one week post-boost. Comparative analysis of the immune responses observed one week post-priming versus the responses detected one week post-boost revealed that the average number of the IFN-γ-secreting CD4(+) T-cells observed in the calves immunized with the CD205 antigen targeting construct increased five-fold, the mean CD4(+) T-cell proliferation increased three-fold, whereas the mean IgG antibody titer increased two hundred-fold. These promising outcomes support testing the protective efficacy of CD205-targeted antigens in the calf model.

Treatment of cattle with DNA-encoded Flt3L and GM-CSF prior to immunization with Theileria parva candidate vaccine antigens induces CD4 and CD8 T cell IFN-γ responses but not CTL responses.

Theileria parva antigens recognized by cytotoxic T lymphocytes (CTLs) are prime vaccine candidates against East Coast fever in cattle. A strategy for enhancing induction of parasite-specific T cell responses by increasing recruitment and activation of dendritic cells (DCs) at the immunization site by administration of bovine Flt3L and GM-CSF prior to inoculation with DNA vaccine constructs and MVA boost was evaluated. Analysis of immune responses showed induction of significant T. parva-specific proliferation, and IFN-γ-secreting CD4(+) and CD8(+) T cell responses in immunized cattle. However, antigen-specific CTLs were not detected. Following lethal challenge, 5/12 immunized cattle survived by day 21, whereas all the negative controls had to be euthanized due to severe disease, indicating a protective effect of the vaccine (p<0.05). The study demonstrated the potential of this technology to elicit significant MHC class II and class I restricted IFN-γ-secreting CD4(+) and CD8(+) T cells to defined vaccine candidate antigens in a natural host, but also underscores the need to improve strategies for eliciting protective CTL responses.

Production and characterization of agonistic monoclonal antibodies against chicken CD40.

CD40 is mainly expressed by professional antigen-presenting cells (APCs). Its ligand, CD40L, is transiently expressed on activated CD4(+) T-cells. CD40-CD40L interactions mediate T-cell help to APCs and provide crucial signals for affinity maturation and B-cell class switching. In mammals, agonistic monoclonal anti-CD40 antibodies (mAbs) mimic the effects of CD40L on APCs, leading to enhanced T-cell priming and expansion, increased antibody production and class switching. In this study, we describe agonistic anti-chicken CD40 mAb 2C5. This mAb detected CD40 on primary chicken B-cells and macrophages, DT40 B-cells, and HD11 macrophages, induced NO synthesis in HD11 macrophages, and stimulated DT40 B-cell proliferation. These observations demonstrated at least partial functional equivalence of 2C5 to chicken CD154. This mAb may therefore constitute a new tool to study the role of CD40 in the chicken immune system, and its agonistic effects suggest that it could also be used as an adjuvant.