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

Introduction: African 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. Methods: In 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). Results: These 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 < 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 < 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. Discussion: Besides 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.

Development and Evaluation of an Enzyme-Linked Immunosorbent Assay Targeting Rabies-Specific IgM and IgG in Human Sera.

Immunity from rabies depends on rabies virus neutralizing antibodies (RVNA) induced after immunization; however, the influence of antibody isotype switching has not been extensively investigated. This has become particularly relevant with changes in World Health Organization (WHO) recommended rabies vaccine regimens that may influence RVNA isotype kinetics, potentially affecting the peak, and longevity, of RVNA immunoglobulin (IgG) levels. We developed rapid and reliable assays for quantifying the anti-rabies IgM/IgG class switch in human serum based on an indirect ELISA technique. The immune response was tracked in ten individuals naïve to the rabies vaccine by quantifying serum titers weekly, from day seven to day 42 post-immunization, using a serum neutralization assay and the ELISA IgM/IgG assays. The average RVNA IU/mL levels were at D0 ≤ 0.1, D7 0.24, D14 8.36, D21 12.84, D28 25.74 and D42 28.68. Levels of specific IgM antibodies to rabies glycoprotein (EU/mL) were higher, on average, at D7, 1.37, and from D14, 5.49, to D21, 6.59. In contrast, average IgG antibodies (EU/mL) predominated from D28, 10.03, to D42, 14.45. We conclude that levels of anti-rabies IgM/IgG at D28 characterize the isotype class switch. These assays, combined with serum neutralization assays, distinguished the RVNA levels in terms of the IgM/IgG responses and are expected to add to the diagnostic repertoire, provide additional information in establishing rabies vaccine regimens, both post- and pre-exposure prophylaxis, and contribute to research efforts.

Robust humoral immune response against rabies virus in rabbits and guinea pigs immunized with plasmid DNA vectors encoding rabies virus glycoproteins - An approach to the production of polyclonal antibody reagents.

DNA-based immunization has been previously shown to be an efficient approach to induce robust immunity against infectious diseases in animals and humans. The advantages of DNA vaccines are simplicity of their construction and production, low cost, high stability, and ability to elicit a full spectrum of immune responses to target antigens. The goals of this study were (i) to assess the antibody immune response to rabies virus glycoproteins (rGPs) in rabbits and guinea pigs after intramuscular immunization with pTargeT and pVAC2-mcs mammalian expression vectors encoding either the wild-type (WT) or codon-optimized (cOPT) rGP genes; and (ii) to prepare in-house rabbit anti-rGP polyclonal antibody reagents suitable for in Single Radial Immunodiffusion (SRID) and Indirect Fluorescent Antibody (IFA) assays. The maximum antibody responses against rabies virus in rabbits and guinea pigs were observed after immunization series with 500 µg/dose of pVAC2-mcs vector encoding either the WT or cOPT rGP genes adjuvanted with Emulsigen-D. No significant difference in the anti-rabies virus neutralizing antibody titers was observed in rabbits immunized with the WT and cOPT rGPs. The in-house rabbit anti-rGP polyclonal antibody reagents reacted comparable to the current reference reagents in SRID and IFA assays. The results of the study demonstrated that the DNA immunization of animals with the WT or cOPT rGPs is a promising approach to either induction of high anti-rabies virus neutralizing antibody titers in vivo or for production of polyclonal antibody reagents against rabies.

Adenovirus-Vectored African Swine Fever Virus pp220 Induces Robust Antibody, IFN-γ, and CTL Responses in Pigs.

African Swine Fever Virus (ASFV) poses a serious threat to the pork industry worldwide; however, there is no safe vaccine or treatment available. The development of an efficacious subunit vaccine will require the identification of protective antigens. The ASFV pp220 polyprotein is essential for virus structural integrity. This polyprotein is processed to generate p5, p34, p14, p37, and p150 individual proteins. Immunization of pigs with a cocktail of adenoviruses expressing the proteins induced significant IgG, IFN-γ-secreting cells, and cytotoxic T lymphocyte responses. Four predicted SLA-I binding nonamer peptides, namely p34161-169, p37859-867, p1501363-1371, and p1501463-1471, recalled strong IFN-γ+ PBMC and splenocyte responses. Notably, peptide p34161-169 was recognized by PBMCs isolated from 7/10 pigs and by splenocytes isolated from 8/10 pigs. Peptides p37859-867 and p1501363-1371 stimulated recall IFN-γ+ responses in PBMCs and splenocytes isolated from 8/10 pigs, whereas peptide p1501463-1471 recalled responses in PBMCs and splenocytes isolated from 7/10 to 9/10 pigs, respectively. The results demonstrate that the pp220 polyprotein contains multiple epitopes that induce robust immune responses in pigs. Importantly, these epitopes are 100% conserved among different ASFV genotypes and were predicted to bind multiple SLA-I alleles. The outcomes suggest that pp220 is a promising candidate for inclusion in a prototype subunit vaccine.