Evaluation of veterinary non-living anthrax vaccine candidates in cattle

Abstract

Anthrax has been the plague of animals and humans for centuries, even though it is primarily a disease of herbivores. Anthrax is caused by the spore-forming bacterium Bacillus anthracis, the virulence of B. anthracis is regulated by two plasmids pXO1 and pXO2. The pXO1 regulates the anthrax toxins by encoding the tripartite proteins, protective antigen (PA), oedema and lethal factors (EF and LF) while the pXO2 regulates the capsule formation by encoding a poly-D-glutamic acid capsule (PDGA) which is immunogenically weak. PA plays a catalytic role to form the anthrax toxins and is the main antigenic component of anthrax. Anthrax is one of the earliest bacterial diseases controlled by vaccination. The B. anthracis Pasteur’s Duplex vaccine and Sterne 34F2 live spore vaccine (SLSV) are live attenuated vaccines which renders them incompatible with accompanying antibiotic treatment in the phase of a disease outbreak. The use of non-living anthrax vaccine (NLAV) which include recombinant PA (rPA), could overcome the shortcomings associated with live attenuated anthrax vaccine by allowing the concurrent use of antibiotics. However, cost-effective production of rPA in a biologically active and up-scalable form is a major challenge. Attempts were made to produce a more cost-effective NLAV that compares with the SLSV. The NLAV used in this study was formulated by improving upon NLAV’s that use rPA, bacillus collagen-like antigen (BclA) and formaldehyde inactivated spores (FIS) in laboratory animals and goats. However, in our study, the NLAV comprised of two formulations of rPA and FIS; purified rPA + FIS (PrPA+FIS) and crude rPA + FIS (CrPA+FIS) both were adjuvanted with a combination of Emulsigen-D®/Alhydrogel®. A two-step vaccination schedule was employed in both vaccination categories and their immunogenicity in vaccinated cattle groups assessed with and without penicillin-G (Pen-G) treatment. Their immunogenicity was compared to cattle groups vaccinated with SLSV with and without Pen-G treatment. An Enzyme-linked immunosorbent assay (ELISA) for immunoglobulins; IgG, IgG1, IgG2 and IgM against rPA and FIS, a toxin neutralisation assay (TNA) and an opsonophagocytic assay were used to assess the immune response in all studies. The level of in vivo protection was determined using a passive mouse protection test. Our findings amongst all groups vaccinated without Pen-G treatment revealed that the polyclonal IgGs, particularly IgG1 and IgM, showed a significant increase across all vaccine groups after the first vaccination. After the second vaccination all IgG subset titres significantly increased across all vaccine groups at week 5 and retained these titres at week 12 when compared to pre-vaccination titres. The toxin neutralisation titres (NT50) among cattle groups vaccinated with the NLAV showed a similar pattern of response but had lower titres, compared with those observed with the ELISA titres. In addition, opsonising antibody responses showed significant spore phagocytosis of 75% (PrPA+FIS), 66% (CrPA+FIS) and 80% (SLSV) following spore opsonisation. In the passive protection test, A/J mice injected with purified polyclonal IgG from cattle vaccinated with PrPA+FIS+Emulsigen-D®/Alhydrogel® and SLSV revealed 73% and 75% protection following challenge with B. anthracis strain 34F2 spores, whereas only 20% protection was recorded among cattle vaccinated with CrPA+FIS+Emulsigen-D®/Alhydrogel®. Furthermore, our findings in the cattle groups vaccinated twice with NLAVs plus Pen-G treatment compared to cattle groups vaccinated twice with SLSV alone, and SLSV plus Pen-G revealed that the IgG titres against rPA for NLAVs plus Pen-G and SLSV without pen-G treatment displayed a significant increase, whereas the titres of the cattle groups immunised with SLSV plus Pen-G treatment was not significant. Contrarily, the IgG titres against FIS revealed a significant antibody titres level among all vaccine groups at week 3 and 5 compared to pre-vaccination titres. The IgM, IgG1 and IgG2 titres against rPA were significant for vaccine groups vaccinated with NLAVs plus Pen-G and SLSV without Pen-G at week 3 and 5 but insignificant for group vaccinated SLSV plus Pen-G. Similar patterns were also revealed for IgM, IgG1 and IgG2 titres against FIS. However, the group vaccinated with SLSV plus Pen-G treatment displayed significant titres against FIS. The toxin neutralisation titres (NT50) was significant among the cattle group vaccinated with NLAVs plus Pen-G and SLSV but not for SLSV plus Pen-G at week 3 and 5 when compared to pre-vaccination titres. Our finding showed the effect of antibiotics on the immunogenicity of SLSV as compared to NLAV and reinforced that cocktails of immune response against both rPA and FIS complement each other thereby conferring better immune response than either rPA or FIS alone. Furthermore, the stimulation of both IgG1 and IgG2 implies a balance between Th1 and Th2 response. The spore opsonising antibodies at all dilution showed a significant opsonisation response among NLAVs plus Pen-G and SLSV without Pen-G vaccine group except at 1:10000 dilution. Our data support the important role of opsonising antibodies in the immune response against infection by presenting spores of B. anthracis to phagocytes. Hence, opsonisation can be adopted as a correlate for the protective immune response against B. anthracis infection. The passive mouse protection test demonstrated significant protection among mice that received polyclonal IgG from SLSV and PrPA+FIS plus Pen-G but not significant for SLSV plus Pen-G and CrPA+FIS plus Pen-G vaccine groups. It is significant to indicate that, in both vaccination categories our findings revealed that there were equivalent to SLSV in protective immune response in cattle vaccinated twice with either the PrPA+FIS or PrPA+FIS plus Pen-G when compared to SLSV without Pen-G treatment. Importantly, our results further confirmed the incompatibility of SLSV with antibiotic treatment. Additionally, NLAV did not show any residual side effects in vaccinated cattle. Finally, our study revealed that NLAV can be used simultaneously with Pen-G antibiotics that will provide long term benefits for future anthrax outbreaks, feedlots and valuable breeding stock.