Development of Adenovirus-vectored Prototype Vaccines for African Swine Fever Virus and Bovine Viral Diarrhea Virus

The objective of this work was to develop adenovirus-vectored prototype vaccines against two pathogens, African Swine Fever Virus (ASFV) and Bovine Viral Diarrhea Virus (BVDV), which cause disease in two major livestock species, swine and cattle respectively. The African Swine Fever Virus is a transboundary animal pathogen that causes a lethal hemorrhagic fever in domestic pigs. Attempts to develop a vaccine for ASFV have failed thus far. This manuscript describes the use of recombinant adenovirus to deliver two unique formulations of ASFV antigens in swine (in two separate in-vivo studies) and the subsequent evaluation of the antigen-specific antibody and cellular responses induced. The robust antigen-specific immune responses observed in both studies are promising and their protective potential will be evaluated in future efficacy studies The Bovine Viral Diarrhea Virus is a globally prevalent pathogen that can cause severe diarrhea, respiratory disease, abortions and sometimes death in calves. Killed and modified live vaccines (MLV) for BVDV have been in use since the 1960s but are not effective due to lack of cross-protection and retention of immunosuppressive characteristics. This thesis also describes the use of the recombinant adenovirus vector to deliver a cocktail of multiple mosaic BVDV antigens in calves followed by the evaluation of protection conferred upon challenge. The prototype vaccine was more immunogenic and cross-protective (based on neutralizing antibodies) than a commercial MLV BVDV vaccine. Regarding protective efficacy, all calves immunized with prototype vaccine cleared the virus within a week post-challenge, whereas one calf that received the MLV vaccine still remained viremic. Future efficacy studies with diverse BVDV strains are required to validate the cross-protective potential of this prototype vaccine

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

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

<div><p>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.</p></div

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

Immunization of pigs with replication-incompetent adenovirus-vectored African swine fever virus multi-antigens induced humoral immune responses but no protection following contact challenge

IntroductionAfrican swine fever virus (ASFV) is a pathogen of great economic importance given that continues to threaten the pork industry worldwide, but there is no safe vaccine or treatment available. Development of a vaccine is feasible as immunization of pigs with some live attenuated ASFV vaccine candidates can confer protection, but safety concerns and virus scalability are challenges that must to be addressed. Identification of protective ASFV antigens is needed to inform the development of efficacious subunit vaccines.MethodsIn this study, replication-incompetent adenovirus-vectored multicistronic ASFV antigen expression constructs that covered nearly 100% of the ASFV proteome were generated and validated using ASFV convalescent serum. Swine were immunized with a cocktail of the expression constructs, designated Ad5-ASFV, alone or formulated with either Montanide ISA-201™ (ASFV-ISA-201) or BioMize® adjuvant (ASFV-BioMize).ResultsThese constructs primed strong B cell responses as judged by anti-pp62-specific IgG responses. Notably, the Ad5-ASFV and the Ad5-ASFV ISA-201, but not the Ad5-ASFV BioMize®, immunogens primed significantly (p &lt; 0.0001) higher anti-pp62-specific IgG responses compared with Ad5-Luciferase formulated with Montanide ISA-201™ adjuvant (Luc-ISA-201). The anti-pp62-specific IgG responses underwent significant (p &lt; 0.0001) recall in all the vaccinees after boosting and the induced antibodies strongly recognized ASFV (Georgia 2007/1)-infected primary swine cells. However, following challenge by contact spreaders, only one pig nearly immunized with the Ad5-ASFV cocktail survived. The survivor had no typical clinical symptoms, but had viral loads and lesions consistent with chronic ASF.DiscussionBesides the limited sample size used, the outcome suggests that in vivo antigen expression, but not the antigen content, might be the limitation of this immunization approach as the replication-incompetent adenovirus does not amplify in vivo to effectively prime and expand protective immunity or directly mimic the gene transcription mechanisms of attenuated ASFV. Addressing the in vivo antigen delivery limitations may yield promising outcomes

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

Persistence of replication-incompetent adenovirus in cattle.

<p>Viable recombinant adenovirus inoculated intradermally is only recoverable within three days. Presence of adenovirus rescued from tissue samples of four steers at defined time points was tracked by immunocytometric analysis of HEK-293A cells. Representative data from one steer is shown: A, D, G, J, and M are positive controls at 24 hr., 48 hr., 72 hr., day 7, and day 21, respectively. B, E, H, and K, are skin biopsies taken from the inoculation sites on the neck of the steers at 24 hr., 48 hr., 72 hr., and day 7, respectively, whereas C, F, I, and L, are cognate control skin biopsies taken concurrently from the flank. N and O are draining lymph node and spleen samples, respectively, collected three weeks post-inoculation.</p

Clinical manifestations post-challenge.

<p>A) Mean rectal temperature fluctuation; and B) Mean change ratios of white blood cell counts in the vaccinated and negative control groups post-challenge. Asterisks denote statistically significant differences as compared to the negative controls. *P<0.05; **P<0.01 and ***P<0.001.</p

Virus isolation from calves on day 7 and day 10 post-challenge.

<p>Virus isolation from calves on day 7 and day 10 post-challenge.</p