Oral rabies vaccination of foxes in Türkiye, 2019-2022.

Background: Rabies in Turkey is maintained by dogs, but following a sustained spill-over, red fox mediated rabies had spread from the Aegean region to the central part of Türkiye. During the past four years from 2019 to 2023 large scale efforts used oral rabies vaccination (ORV) to control rabies in red foxes. Here, we present the results of the largest ORV campaign on the Asian continent. Methods: ORV campaigns were carried out twice a year in spring and autumn with a targeted bait density of 20-23 baits/km2. Monitoring of ORV campaigns included the GIS-based analyses of bait distribution, the assessment of bait uptake through biomarker detection and the determination of seroconversion (sero-positivity in ELISA) in the target species collected within the vaccination area. For determination of fox rabies incidence in vaccination areas as the main indicator of the performance of the ORV campaigns, epidemiological data was obtained from the national passive surveillance program. Results: Aerial bait distribution was highly accurate, with >99 % of baits being recorded from targeted zones, thus meeting the desired bait densities. Although the overall bait uptake (28.1 %; 95 %CI: 23.2-32.8) and seroprevalance (36.3 %; 95 %CI: 30.0-43.2) were low, rabies incidence drastically decreased in ORV areas and rabies was eliminated from western and central parts of Turkey, with no reported cases in foxes from ORV areas in 2022 and 2023. Conclusions: A large-scale ORV campaign against fox rabies using high quality vaccine baits and the GIS-aided and monitored bait distribution was able to control fox mediated rabies in the western and central parts of Türkiye. Rabies control both in dogs and foxes should be expanded to cover also the eastern parts of Türkiye, to become eventually rabies free.

Evaluation of Bait Acceptance and Immune Response in Local Dogs during an Oral Rabies Vaccination Field Study in Morocco.

The objective of this study was to evaluate the bait preference of three selected bait types by local dogs and the induced immunogenicity of the oral rabies vaccine strain SPBN GASGAS in Morocco. The vaccine strain, combined with different bait types, has been tested in many different settings, but not yet in northern Africa. Overall, bait consumption and preference were similar in other studies using the same materials (bait type and sachet). The intestine bait had the highest acceptance rate (97.6%, 95%CI: 87.4-99.9), followed by the egg bait (83.0%, 95%CI: 69.2-92.4). Only 52% (95%CI: 37.4-66.3) of the dogs showed an interest in the fish meal bait. However, considering the successful release of the contents of the sachet (blue-dyed water) into the oral cavity, the egg bait (65.7%, 95%CI: 47.8-80.9) scored better than the intestine bait (51.7%, 95%CI: 32.5-70.6). The dogs selected for the immunogenicity study were offered the egg bait containing a sachet filled with SPBN GASGAS (3.0 mL, 107.5 FFU/mL) or were given the same dose by direct oral administration (d.o.a.). In addition, several dogs were vaccinated by the parenteral route (s.c.) using a commercially available inactivated rabies vaccine. Unfortunately, due to the COVID-19 pandemic and subsequent travel restrictions, it was not possible to collect blood samples directly after vaccination. The blood samples were collected pre-vaccination and on five occasions between 450 and 1088 days post vaccination. The seroconversion rate, as determined for rabies-virus-neutralizing antibodies by the FAVN test, was significantly lower than that found for binding antibodies, as determined by ELISA, for all blood samples collected post vaccination. No treatment effect (bait, d.o.a., s.c.) could be seen in the seroconversion rate. At 15 months post vaccination, 84.2% of the dogs offered vaccine bait still tested sero-positive in ELISA. Only after 3 years was a clear drop in the seroconversion rate observed in all three treatment groups. This study confirms the long-term immunogenicity of the oral rabies vaccine SPBN GASGAS in dogs under field conditions.

Improved stability and specificity of baits for oral administration of substances to wild boar.

Oral vaccination is one of the most effective interventions for disease control in wildlife. As a result of the recent global reemergence of African swine fever and ongoing classical swine fever and animal tuberculosis, oral vaccination of Eurasian wild boar (Sus scrofa) receives increased interest. Several baits for wild boar and feral pigs have been described, but developing more stable and personalized formulations is important. This paper proposes a new bait formulation primarily composed of corn flour, piglet feed, sugar, and honey as a binder to obtain improved elasticity. The bait consists of a matrix with no protective coats, has a hemispherical shape (ø 3.4 ×1.6 cm), and displays an anise aroma and blue color. The color and aroma did not affect bait choice by wild boar, while bait coloring contributed to avoid consumption by non-target species (corvids). Baits with the new formulation were significantly more resistant to humidity and high temperatures than previous versions. Simulations suggest that baits with the new formulation are elastic enough to resist impacts from a maximum altitude of 750 m. Thus, the new bait prototype solves several problems of previous bait formulations while keeping a format that can be selectively consumed by piglets and adult wild boar.

Evaluation of the efficacy of MONTANIDE™ GR01, a new adjuvant for feed-based vaccines, on the immune response and protection against Streptococcus agalactiae in oral vaccinated Nile tilapia (Oreochromis niloticus) under laboratory and on-farm conditions.

Streptococcosis, an emerging infectious disease caused by Streptococcus agalactiae, has had adverse effects on farmed tilapia. Several vaccines have been developed to prevent this disease and induce a specific immune response against S. agalactiae infection. In this study the use of MONTANIDE™ GR01, a new adjuvant for oral vaccination, was optimized for use in tilapia under laboratory and field studies. In the laboratory trial the immune response and protective efficacy of two doses of MONTANIDE™ GR01, 20 % (w/w) and 2 % (w/w), included into the feed-based adjuvanted vaccines were assessed comparatively. Following immunization, the innate immune parameters studied in serum, including lysozyme, myeloperoxidase, catalase and glutathione peroxidase activity, were all increased significantly. Furthermore, specific IgM antibodies against S. agalactiae were induced significantly in serum post-vaccination, with higher levels observed in both groups that received the feed-based adjuvanted vaccine. Under both injection and immersion challenge conditions, the relative percent survival for the feed-based adjuvanted vaccine groups ranged from 78 % to 84 %. Following use of the low dose concentration of MONTANIDE™ GR01 for oral vaccination of tilapia in cage culture systems, several innate immune parameters were effectively enhanced in the immunized fish. Similarly, the levels of specific IgM antibodies in the serum of feed-based vaccinated fish were significantly enhanced, reaching their highest levels 2-5 months post-vaccination. Cytokines associated with innate and adaptive immunity were also examined, and the expression levels of several genes showed significant up-regulation. This indicates that both cellular and humoral immune responses were induced by the feed-based adjuvanted vaccine. The economic impact of a feed-based adjuvanted vaccine was examined following vaccination, considering the growth performance and feed utilization of the fish. It was found that the Economic Performance Index and Economic Conversion Ratio were unaffected by vaccination, further demonstrating that there are no negative impacts associated with administering a feed-based vaccine to fish. In conclusion, the data from this study indicate that MONTANIDE™ GR01 is a highly valuable adjuvant for oral vaccination, as demonstrated by its ability to induce a strong immune response and effectively prevent streptococcal disease in Nile tilapia.

Mechanism of Action of Oral Salmonella-Based Vaccine to Prevent and Reverse Type 1 Diabetes in NOD Mice.

A combination therapy of preproinsulin (PPI) and immunomodulators (TGFß+IL10) orally delivered via genetically modified Salmonella and anti-CD3 promoted glucose balance in in NOD mice with recent onset diabetes. The Salmonella bacteria were modified to express the diabetes-associated antigen PPI controlled by a bacterial promoter in conjunction with over-expressed immunomodulating molecules. The possible mechanisms of action of this vaccine to limit autoimmune diabetes remained undefined. In mice, the vaccine prevented and reversed ongoing diabetes. The vaccine-mediated beneficial effects were associated with increased numbers of antigen-specific CD4+CD25+Foxp3+ Tregs, CD4+CD49b+LAG3+ Tr1-cells, and tolerogenic dendritic-cells (tol-DCs) in the spleens and lymphatic organs of treated mice. Despite this, the immune response to Salmonella infection was not altered. Furthermore, the vaccine effects were associated with a reduction in islet-infiltrating lymphocytes and an increase in the islet beta-cell mass. This was associated with increased serum levels of the tolerogenic cytokines (IL10, IL2, and IL13) and chemokine ligand 2 (CCL2) and decreased levels of inflammatory cytokines (IFNγ, GM-CSF, IL6, IL12, and TNFα) and chemokines (CXCL1, CXCL2, and CXCL5). Overall, the data suggest that the Salmonella-based vaccine modulates the immune response, reduces inflammation, and promotes tolerance specifically to an antigen involved in autoimmune diabetes.

A Global Perspective on Oral Vaccination of Wildlife against Rabies.

The long-term mitigation of human-domestic animal-wildlife conflicts is complex and difficult. Over the last 50 yr, the primary biomedical concepts and actualized collaborative global field applications of oral rabies vaccination to wildlife serve as one dramatic example that revolutionized the field of infectious disease management of free-ranging animals. Oral vaccination of wildlife occurred in diverse locales within Africa, Eurasia, the Middle East, and North America. Although rabies is not a candidate for eradication, over a billion doses of vaccine-laden baits distributed strategically by hand, at baiting stations, or via aircraft, resulted in widespread disease prevention, control, or local disease elimination among mesocarnivores. Pure, potent, safe, and efficacious vaccines consisted of either modified-live, highly attenuated, or recombinant viruses contained within attractive, edible baits. Since the late 1970s, major free-ranging target species have included coyotes (Canis latrans), foxes (Urocyon cinereoargenteus; Vulpes vulpes), jackals (Canis aureus; Lupulella mesomelas), raccoons (Procyon lotor), raccoon dogs (Nyctereutes procyonoides), and skunks (Mephitis mephitis). Operational progress has occurred in all but the latter species. Programmatic evaluations of oral rabies vaccination success have included: demonstration of biomarkers incorporated within vaccine-laden baits in target species as representative of bait contact; serological measurement of the induction of specific rabies virus neutralizing antibodies, indicative of an immune response to vaccine; and most importantly, the decreasing detection of rabies virus antigens in the brains of collected animals via enhanced laboratory-based surveillance, as evidence of management impact. Although often conceived mistakenly as a panacea, such cost-effective technology applied to free-ranging wildlife represents a real-world, One Health application benefiting agriculture, conservation biology, and public health. Based upon lessons learned with oral rabies vaccination of mesocarnivores, opportunities for future extension to other taxa and additional diseases will have far-reaching, transdisciplinary benefits.

Effects of Dietary Supplementation of Essential Oils, Lysozyme, and Vitamins' Blend on Layer Hen Performance, Viral Vaccinal Response, and Egg Quality Characteristics.

Maintaining respiratory tract health is crucial for layers, impacting gut health, laying performance, and egg quality. Viral diseases and standard vaccinations can compromise tracheal epithelium function, leading to oxidative stress. This study assessed the impact of a blend of feed additives, predominantly lysozyme (L), essential oils (EO), and vitamins (VIT) (referred to as L + EO + VIT), on young layers during an oral vaccination schedule. The supplementation significantly enhanced antibody titers for Newcastle Disease Virus (NDV) and Infectious Bronchitis Virus (IBV) after vaccination, trachea functionality and intestinal health in the jejunum, increased egg production, and exhibited a trend toward higher egg weight. Although feed intake showed no significant difference, egg quality remained consistent across experimental groups. Moreover, L + EO + VIT supplementation elevated total phenolic content in eggs, improving oxidative stability in both fresh and stored eggs, particularly under iron-induced oxidation. Notably, it substantially reduced yolk lipid peroxidation and albumen protein carbonyls. In conclusion, water supplementation with L + EO + VIT may enhance humoral immune response to IBV and NDV, positively impacting hen productivity. These findings indicate improved tracheal function and enhanced oxidative stability, emphasizing the potential of this blend in promoting overall health and performance in layers.

Retrospective analysis of sero-prevalence and bait uptake estimations in foxes after oral rabies vaccination programmes at European level: Lessons learned and paths forward.

Rabies caused by the Classical Rabies Virus (Lyssavirus rabies abbreviated RABV) in the European Union has been close to elimination mainly thanks to Oral Rabies Vaccination (ORV) campaigns targeting wildlife (primarily red foxes). ORV programmes co-financed by the European Commission include a monitoring-component to assess the effectiveness of the ORV campaigns at national level. This assessment is performed by a random collection of red foxes in the vaccinated areas with control of antibodies presence by serological analysis and control of bait uptake by detection of biomarkers (tetracycline incorporated into the baits) in the bones and teeth. ORV programmes aim to a vaccine coverage high enough to immunize (ideally) 70 % of the reservoir population to control the spread of the disease. European Union (EU) programmes that led to almost elimination of rabies on the territory have been traditionally found to have a bait uptake average of 70 % (EU countries; 2010-2020 period) while the seroconversion data showed an average level of 40 % (EU countries; 2010-2020 period). To better understand variations of these indicators, a study was been set up to evaluate the impact of several variables (linked to the vaccination programme itself and linked to environmental conditions) on the bait uptake and the seroconversion rate. Thus, pooling data from several countries provides more powerful statistics and the highest probability of detecting trends. Results of this study advocate the use of a single serological test across the EU since data variation due to the type of test used was higher than variations due to field factors, making the interpretation of monitoring results at EU level challenging. In addition, the results indicates a negative correlation between bait uptake and maximum temperatures reached during ORV campaigns questioning the potential impact of climatic change and associated increase of temperatures on the ORV programmes efficiency. Several hypotheses requesting additional investigation are drawn and discussed in this paper.

Long-term immunity induced by SPBN GASGAS in orally vaccinated dogs is non-inferior to inactivated rabies vaccines.

In a long-term immunogenicity study (1100 days post vaccination) in local Thai dogs the immune response of the oral rabies vaccine SPBN GASGAS was compared to those elicited by a commercial inactivated vaccine using immunobridging. Based on the detection of rabies virus binding (rVBA) and rabies virus neutralizing antibodies (rVNA) as measured by ELISA and Rapid Fluorescent Focus Inhibition Test (RFFIT) the long-term immune response in dogs vaccinated orally with the SPBNA GASGAS strain of rabies vaccine in a bait was non-inferior to a conventional inactivated rabies vaccine. The outcome of this study supports extending the originally claimed duration of immunity (DOI) of SPBN GASGAS after oral vaccination for dogs from 6 to 30 months.

Is Oral Vaccination against Escherichia coli Influenced by Zinc Oxide?

BACKGROUND: Although zinc oxide has been banned at therapeutic doses in the EU, its use is still legal in most countries with industrial pig farming. This compound has been shown to be very effective in preventing E. coli-related diseases. However, another strategy used to control this pathogen is vaccination, administered parenterally or orally. Oral vaccines contain live strains, with F4 and F18 binding factors. Since zinc oxide prevents E. coli adhesion, it is hypothesised that its presence at therapeutic doses (2500 ppm) may alter the immune response and the protection of intestinal integrity derived from the vaccination of animals. METHODS: A group of piglets were orally vaccinated at weaning and divided into two subgroups; one group was fed a feed containing 2500 ppm zinc oxide (V + ZnO) for the first 15 days post-vaccination (dpv) and the other was not (V). Faeces were sampled from the animals at 6, 8, 11, 13, and 15 dpv. Unvaccinated animals without ZnO in their feed (Neg) were sampled simultaneously and, on day 15 post-vaccination, were also compared with a group of unvaccinated animals with ZnO in their feed (ZnO). RESULTS: Differences were found in E. coli excretion, with less quantification in the V + ZnO group, and a significant increase in secretory IgA in the V group at 8 dpv, which later equalised with that of the V + ZnO group. There was also some difference in IFNα, IFNγ, IL1α, ILß, and TNFα gene expression when comparing both vaccinated groups (p < 0.05). However, there was no difference in gene expression for the tight junction (TJ) proteins responsible for intestinal integrity. CONCLUSIONS: Although some differences in the excretion of the vaccine strain were found when comparing both vaccinated groups, there are no remarkable differences in immune stimulation or soluble IgA production when comparing animals orally vaccinated against E. coli in combination with the presence or absence of ZnO in their feed. We can conclude that the immune response produced is very similar in both groups.

Optimising rabies vaccination of dogs in India.

Dog vaccination is the key to controlling rabies in human populations. However, in countries like India, with large free-roaming dog populations, vaccination strategies that rely only on parenteral vaccines are unlikely to be either feasible or successful. Oral rabies vaccines could be used to reach these dogs. We use cost estimates for an Indian city and linear optimisation to find the most cost-effective vaccination strategies. We show that an oral bait handout method for dogs that are never confined can reduce the per dog costs of vaccination and increase vaccine coverage. This finding holds even when baits cost up to 10x the price of parenteral vaccines, if there is a large dog population or proportion of dogs that are never confined. We suggest that oral rabies vaccine baits will be part of the most cost-effective strategies to eliminate human deaths from dog-mediated rabies by 2030.

Targeted delivery of oral vaccine antigens to aminopeptidase N protects pigs against pathogenic E. coli challenge infection.

Oral subunit vaccines are an interesting alternative strategy to traditional live-attenuated or inactivated vaccines for conferring protection against gut pathogens. Despite being safer and more cost-effective, the development of oral subunit vaccines remains challenging due to barriers imposed by the gastrointestinal tract, such as digestive enzymes, a tolerogenic immune environment and the inability of larger proteins to cross the epithelial barrier. Recent advances have focused on overcoming these barriers by using potent mucosal adjuvants or pH-responsive delivery vehicles to protect antigens from degradation and promote their release in the intestinal lumen. A promising approach to allow vaccine antigens to pass the epithelial barrier is by their targeting towards aminopeptidase N (APN; CD13), an abundant membrane protein present on small intestinal enterocytes. APN is a peptidase involved in digestion, but also a receptor for several enteric pathogens. In addition, upon antibody-mediated crosslinking, APN facilitated the transport of antibody-antigen fusion constructs across the gut epithelium. This epithelial transport resulted in antigen-specific immune responses. Here, we present evidence that oral administration of APN-specific antibody-antigen fusion constructs comprising the porcine IgA Fc-domain and the FedF tipadhesin of F18-fimbriated E. coli elicited both mucosal and systemic immune responses and provided at least partial protection to piglets against a subsequent challenge infection with an F18-fimbriated STEC strain. Altogether, these findings will contribute to the further development of new oral subunit vaccines and provide a first proof-of-concept for the protective efficacy of APN-targeted vaccine antigens.

Oral delivery of a Streptococcus agalactiae vaccine to Nile tilapia (Oreochromis niloticus) using a novel cationic-based nanoemulsion containing bile salts.

Streptococcus agalactiae is one of Thailand's most important pathogens in tilapia aquaculture. Vaccination is a very effective method for protecting fish against disease in aquaculture. Oral vaccination is an interesting route for vaccine delivery as it mimics the pathogenesis of S. agalactiae and provides convenient administration for mass vaccination of fish. Moreover, gut mucosal immunity is associated with a mucus layer on the gastrointestinal tract. Therefore, this study aimed to develop a novel cationic-based nanoemulsion vaccine containing bile salts (NEB) coated by chitosan (CS) and determined its physicochemical characterization, morphology, in vitro mucoadhesive property, permeability, and acid-base tolerance. In addition, the efficacy of NEB-CS as an oral vaccination for Nile tilapia was evaluated in order to investigate the innate immune response and protection against S. agalactiae. The groups of fish consisted of: (1) deionized water as a non-vaccinated control (Control); (2) an inactivated vaccine formulated from formalin-killed bacteria (IB); and (3) a novel cationic-based nanoemulsion vaccine containing bile salts (NEB) coated by chitosan (CS). The control, IB, and NEB-CS were incorporated into commercial feed pellets and fed to Nile tilapia. In addition, we evaluated the serum bactericidal activity (SBA) for 14 days post-vaccination (dpv) and protective efficacy for 10 days post-challenge, respectively. The mucoadhesiveness, permeability, and absorption within the tilapia intestine were also assessed in vivo. The NEB-CS vaccine appeared spherical, with the nanoparticles having a size of 454.37 nm and a positive charge (+47.6 mV). The NEB-CS vaccine had higher levels of mucoadhesiveness and permeability than the NEB (p < 0.05). The relative percent survival (RPS) of IB and NEB-CS, when administered orally to fish, was 48% and 96%, respectively. Enhanced SBA was noted in the NEB-CS and IB vaccine groups compared to the control group. The results demonstrate that a feed-based NEB-CS can improve the mucoadhesiveness, permeability, and protective efficacy of the vaccine, and appear to be a promising approach to protecting tilapia in aquaculture against streptococcosis.

Emergency response using oral rabies vaccination of dogs -field data from Namibia demonstrate high efficiency.

Dog-mediated rabies is responsible for tens of thousands of human deaths annually, and in resource-constrained settings, vaccinating dogs to control the disease at source remains challenging for various reasons. Currently, rabies elimination efforts rely on mass dog vaccination by the parenteral route. While oral rabies vaccination (ORV) of dogs is primarily considered a tool to increase herd immunity, particularly by targeting free-roaming and stray dogs, here, we are showcasing an ORV-only approach as an emergency response model. Using a third-generation vaccine and a standardized egg-flavored bait, we assessed the effectiveness and vaccination under field conditions in the Zambezi region of Namibia. During this trial, with four teams and within four working days, 3097 dogs were offered a bait, of which 88,0% were considered vaccinated. Teams managed to vaccinate, on average, over 20 dogs/h, despite using a door-to-door vaccination approach. The favorable results both in terms of bait acceptance and successful vaccination as well as field applicability and effectiveness further support the great potential of ORV in dog rabies control programmes.

Maternal Breast Milk Secretor Phenotype Does Not Affect Infant Susceptibility to Rotavirus Diarrhea.

Breast milk secretor status is associated with antibody seroconversion to oral rotavirus vaccination. Here, we were unable to detect a similar impact on risk of infant rotavirus diarrhea or vaccine efficacy through 2 years of life, underscoring limitations of immunogenicity assessment alone in evaluation of oral rotavirus vaccine response.

Surface display of major capsid protein on Bacillus subtilis spores against largemouth bass virus (LMBV) for oral administration.

Largemouth bass virus (LMBV) infections resulting in enormous loss are becoming an increasing problem in the largemouth bass industry. Oral vaccination is considered to be an effective and economical measure because of the advantages of non-invasion, no size limitation, lower cost and easily-operated. Based on Bacillus subtilis (B. subtilis) spores, this study successfully constructed the CotC-LMBV recombinant B. subtilis spores and its protective efficacy and immune responses were evaluated. After challenged, the survival rate of largemouth bass orally vaccinated with CotC-LMBV spores was 53.3% and the relative percent survival (RPS) was 45.0% compared to the PBS group. In addition, the specific IgM level in serum in the CotC-LMBV group was significantly higher than in the control groups. In the spleen, the immune-related genes expression detected by quantitative real-time PCR (qRT-PCR) exhibited an increasing trend in different degrees in the CotC-LMBV group, suggesting that innate and adaptive immune responses were activated. This study indicated that oral administration of CotC-LMBV recombinant spores could stimulate an effective immune response and enhance fish immunity against LMBV infection. Therefore, oral vaccination could be an effective approach for the prevention of largemouth bass virus disease.

Efficacy of Oral Rabies Vaccine Baits Containing SPBN GASGAS in Domestic Dogs According to International Standards.

(1) Background: The oral vaccination of free-roaming dogs against rabies has been developed as a promising complementary tool for mass dog vaccination. However, no oral rabies vaccine has provided efficacy data in dogs according to international standards. (2) Methods: To test the immunogenicity and efficacy of the third-generation oral rabies virus vaccine strain, SPBN GASGAS, in domestic dogs, dogs were offered an egg-flavoured bait containing 3.0 mL of the vaccine (107.5 FFU/mL) or a placebo egg-flavoured bait. Subsequently, these 25 vaccinated and 10 control animals were challenged approximately 6 months later with a dog rabies virus isolate. Blood samples were collected at different time points postvaccination and examined by ELISA and RFFIT. (3) Results: All but 1 of the 25 vaccinated dogs survived the challenge infection; meanwhile, all 10 control dogs succumbed to rabies. The serology results showed that all 25 vaccinated dogs seroconverted in ELISA (>40% PB); meanwhile, only 13 of the 25 vaccinated dogs tested seropositive ≥ 0.5 IU/mL) in RFFIT. (4) Conclusions: The SPBN GASGAS rabies virus vaccine meets the efficacy requirements for live oral rabies vaccines as laid down by the European Pharmacopoeia and the WOAH Terrestrial Manual. SPBN GASGAS already fulfilled the safety requirements for oral rabies vaccines targeted at dogs. Hence, the egg-flavoured bait containing SPBN GASGAS is the first oral vaccine bait that complies with WOAH recommendations for the intended use of oral vaccination of free-roaming dogs against rabies.

Spray Drying Is a Viable Technology for the Preservation of Recombinant Proteins in Microalgae.

Microalgae are promising host organisms for the production of encapsulated recombinant proteins such as vaccines. However, bottlenecks in bioprocess development, such as the drying stage, need to be addressed to ensure feasibility at scale. In this study, we investigated the potential of spray drying to produce a recombinant vaccine in microalgae. A transformant line of Chlamydomonas reinhardtii carrying a subunit vaccine against salmonid alphavirus was created via chloroplast engineering. The integrity of the recombinant protein after spray drying and its stability after 27 months storage at -80 °C, +4 °C and room temperature were assessed by immunoblotting. The protein withstood spray drying without significant losses. Long-term storage at +4 °C and room temperature resulted in 50% and 92% degradation, respectively. Optimizing spray drying and storage conditions should minimize degradation and favour short-term storage at positive temperatures. Using data on yield and productivity, the economics of spray drying- and freeze drying-based bioprocesses were compared. The drying stage corresponded to 41% of the total production cost. Process optimization, genetic engineering and new market strategies were identified as potential targets for cost reduction. Overall, this study successfully demonstrates the suitability of spray drying as a process option for recombinant protein production in microalgae at the industrial scale.

Oral Vaccination of Recombinant Saccharomyces cerevisiae Expressing ORF132 Induces Protective Immunity against Cyprinid Herpesvirus-2.

Cyprinid herpesvirus 2 (CyHV-2) is the etiological agent of herpesviral hematopoietic necrosis (HVHN) disease, which causes serious economic losses in the crucian carp culture industry. In this study, by displaying ORF132 on the surface of Saccharomyces cerevisiae cells (named EBY100/pYD1-ORF132), we evaluated the protective efficacy of oral administration against CyHV-2 infection. Intense innate and adaptive immune responses were evoked in both mucosal and systemic tissues after oral vaccination with EBY100/pYD1-ORF132. Importantly, oral vaccination provided significant protection for crucian carp post CyHV-2 infection, resulting in a relative percent survival (RPS) of 64%. In addition, oral administration suppressed the virus load and relieved histological damage in selected tissues. Our results indicated that surface-displayed ORF132 on S. cerevisiae could be used as potential oral vaccine against CyHV-2 infection.

Oral delivery of bi-autoantigens by bacterium-like particles (BLPs) against autoimmune diabetes in NOD mice.

Induction of oral tolerance by vaccination with type 1 diabetes mellitus (T1DM)-associated autoantigens exhibits great potential in preventing and treating this autoimmune disease. However, antigen degradation in the gastrointestinal tract (GIT) limits the delivery efficiency of oral antigens. Previously, bacterium-like particles (BLPs) have been used to deliver a single-chain insulin (SCI-59) analog (BLPs-SCI-59) or the intracellular domain of insulinoma-associated protein 2 (IA-2ic) (BLPs-IA-2ic). Both monovalent BLPs vaccines can suppress T1DM in NOD mice by stimulating the corresponding antigen-specific oral tolerance, respectively. Here, we constructed two bivalent BLPs vaccines which simultaneously deliver SCI-59 and IA-2ic (Bivalent vaccine-mix or Bivalent vaccine-SA), and evaluated whether there is an additive beneficial effect on tolerance induction and suppression of T1DM by treatment with BLPs-delivered bi-autoantigens. Compared to the monovalent BLPs vaccines, oral administration of the Bivalent vaccine-mix could significantly reduce morbidity and mortality in T1DM. Treatment with the bivalent BLPs vaccines (especially Bivalent vaccine-mix) endowed the mice with a stronger ability to regulate blood glucose and protect the integrity and function of pancreatic islets than the monovalent BLPs vaccines treatment. This additive effect of BLPs-delivered bi-autoantigens on T1DM prevention may be related to that SCI-59- and IA-2-specific Th2-like immune responses could be induced, which was more beneficial for the correction of Th1/Th2 imbalance. In addition, more CD4+CD25+Foxp3+ regulatory T cells (Tregs) were induced by treatment with the bivalent BLPs vaccines than did the monovalent BLPs vaccines. Therefore, multiple autoantigens delivered by BLPs maybe a promising strategy to prevent T1DM by efficiently inducing antigen-specific immune tolerance.