Study on Modulation of Vaccine Immune Response with Probiotic Supplementation in Animal Models

Abstract

Background: Probiotics besides several health benefits on the host, are also consideredas growth promoters on a large scale to improve the productivity of many animal species for human consumption. New Zealand white rabbits are the ideal laboratory animals for their use as experimental models in research and bio-medicine due to economic feasibility. This studyaimed to evaluate the potential adjuvant-like properties of three strains of commercial probiotics on the growth performance (biochemical and hematological parameters and body weight of rabbits) and humoral response against rabies vaccination. Secondly by further secondary hyper-immunization strategy using ID route of immunization, higher antibody titers could be obtained which can further be purified with the use of caprylic acid-based fractionation techniques to produce high-quality anti-sera from animal plasma. The modern purification techniques are simple, practical, and cost effective with the potential to alleviate Pakistan's chronic shortage of rabies vaccine and antiserum in the local market. Methodology: New Zealand white rabbits were chosen as animal models to study the effects of antibiotics, the mineral zinc, and organically carrot dietary supplements on general growth and immunological response to the rabies vaccine. The rabbits were immunised using concentrated rabies vaccine "CCRV" using Thai red crosses intradermal "TRC-ID" immunization. The feed supplementation started one week prior to vaccination till the completion of the experiment at day 60th. The primary outcome was to achieve improved sero-conversion with simple dietary interventions when contrasted with a control group in testing groupings. Results: Our results mainly showed a comparison of the humoral immunological reaction as specific titre of antibodies against vaccination among different groups with dietary interventions. The experimental probiotic groups (G1-G6) displayed high mean titre values of 3.506-3.896, 3.804-3.962 and 4.144-4.336 EU/L at day 14, 35 and 60 respectively. While control group C had lowest titre value with 3.089, 3.505 and 3.7462 EU/L at 14th, 35th and 60th day respectively. xix The optimal results were achieved by introducing caprylic acid solution into plasma,at pH 5.8, and 5% final caprylic acid concentration . Subsequently, the mixture was stirred for an additional 60 minutes and passed through different filters. This downstream production method involves a single precipitation stage and dialysis. The comparison of caprylic acid-based fractionation and classical ammonium sulphate purification techniques revealed that the former produces a final product with superior yield and quality, meeting the recommended criteria for rabies antiserum. Conclusion: Our research has determined that implementing low-cost dietary interventions, such as probiotics and vitamins, in conjunction with improved immunization strategies can effectively decrease the overall expense of rabies vaccinations. The use of this technology presents a promising solution for locally producing anti-sera products, offering both efficiency and simplicity. Therefore, the incorporation of probiotics and vitamin-rich diets for rabbits significantly influences their overall growth and immunity levels– an approach that can be applied successfully to produce animal-derived rabies antiserum products. The study aimed to discover further economical strategies to produce animal-derived Rabies immunoglobulins/antiserum (RIGs). Adopting intradermal vaccine administration schedules can reduce vaccine shortages, increase treatment affordability, and provide equitable access to rabies PEP. The hyper-immunization approach was also utilized to enhance PAb production in animals. To purify the final product, the caprylic acid fractionation technique was implemented to improve downstream processing, thus yielding a purified immunoglobulin fraction that surpasses classical salt precipitation techniques. These cost-effective strategies could prove advantageous to commercial manufacturers producing large-scale rabies immunoglobulins from animal plasma in developing nations. These approaches are particularly relevant for countries with lower to moderate incomes where more efficient measures are necessary to alleviate human suffering caused by rabies. With efforts already underway, adopting various upstream and downstream strategies can assist developing countries like Pakistan in achieving goal of eliminating dog-mediated rabies deaths and reducing the global burden of rabies by 2030. xx RATIONALE/AUTHOR’SSUMMARY Rabies is the oldest recorded and most fatal of all infectious diseases, yet this zoonotic disease remains a neglected considering an elevated prevalence of bites from dogs, it is a disregarded indigenous illness in Pakistan in both rural and urban areas. Unfortunately, there are insufficient epidemiological statistics on the disease, and no proper control program exists in Pakistan at national level. The National Health Management Information System (NHMIS) recorded roughly more than ninety-seven thousand bites from dogs incidences from three main cities in Pakistan in 2016 with estimated (59.7%) cases from Karachi, 13% from Peshawar and 11% from Hyderabad cities (Ahmad et al., 2016). However timely intervention including proper prevention, control of canine rabies with immediate treatment can save human lives (Kumar & Bakhru, 2022). In locations where dog attacks are a particularly frequent and biggest cause of populace misery, rabies vaccination constitutes one of the most critical prophylactic interventions. However,the shortage of vaccine and rabies immunoglobulins (RIG) causes approximately 2000-5000 deaths in Pakistan (HRI, 2023). This high incidence of human rabies is mainly coupled with a lack of awareness about the disease complications, shortage of locally produced vaccines and anti-sera and the overall costs of imported cell culture vaccines and imported human rabies immunoglobulins (HRIG) (Table-7). For the strengthening of indigenous antiserum production, there is dire need to develop cost-effective and novel immune-enhancing strategies for optimization of production methodologies at different manufacturing stages, to get better product yield at the end. In this study simple dietary interventions were applied to enhance the NZW rabbits' humorous immunological reactions to rabies immunization. According to the findings of this investigation, treatment of probiotics and vitamin A rich diet along with rabies vaccination of animals is a simple, cost-effective and unique approach for production of animal derived poly-clonal antibodies (anti-sera). Moreover, the new caprylic acid fractionation method described for protein purification from plasma provided better recovery yield of purified immunoglobulin fraction in a short period of time. xx