Novel particulate vaccine candidates recombinantly produced by pathogenic and nonpathogenic bacterial hosts : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Manawatu, New Zealand.

Polyhydroxyalkanoates (PHAs) are biopolyesters synthesized as small spherical cytoplasmic inclusion bodies by a range of bacteria. Recently, PHA beads have been investigated for use as a vaccine delivery platform by using engineered heterologous production hosts that allowed the efficient display of vaccine candidate antigens on the beads surface and were found to greatly improve immunogenicity of the displayed antigens. However, like other subunit vaccines, these antigen-displaying (vaccine) PHA beads only provide a limited repertoire of antigens. In this thesis we investigate the idea of directly utilizing the disease causative pathogen or model organism to produce vaccine PHA beads with a large antigenic repertoire. These beads are hypothesized to have the potential to induce greater protective immunity compared to production of the same PHA bead in a heterologous production host. This concept was exemplified with Pseudomonas aeruginosa and Mycobacterium tuberculosis as model human pathogens. For P. aeruginosa we describe the engineering of this bacterium to promote PHA and Psl (polysaccharide) production. This represents a new mode of functional display for the engineering, production, and validation of a novel OprI/F-AlgE fusion antigen-displayed on PHA beads. For the disease tuberculosis we investigated the use of nonpathogenic M. smegmatis as a model organism for M. tuberculosis. We described the bioengineering, production, and validation of Ag85AESAT- 6 displayed on PHA beads produced in M. smegmatis. Here we showed that both organisms were harnessed to produce custom-made PHA beads for use as particulate subunit vaccines that carried copurifying pathogen-derived proteins as a large antigenic repertoire and the ability of these vaccine PHA beads to generate a protective immune response. This novel bioengineering concept of particulate subunit vaccine production could be applied to a range of pathogens naturally producing PHA inclusions for developing efficacious subunit vaccines for infectious diseases

Reducing Campylobacter jejuni colonization in broiler chickens by in-feed supplementation with hyperimmune egg yolk antibodies

Campylobacter infections sourced mainly to poultry products, are the most important bacterial foodborne zoonoses worldwide. No effective measures to control these infections in broiler production exist to date. Here, we used passive immunization with hyperimmune egg yolks to confer broad protection of broilers against Campylobacter infection. Two novel vaccines, a bacterin of thirteen Campylobacter jejuni (C. jejuni) and C. coli strains and a subunit vaccine of six immunodominant Campylobacter antigens, were used for the immunization of layers, resulting in high and prolonged levels of specific immunoglobulinY (IgY) in the hens' yolks. In the first in vivo trial, yolks (sham, bacterin or subunit vaccine derived) were administered prophylactically in the broiler feed. Both the bacterin and subunit vaccine-induced IgY significantly reduced the number of Campylobacter-colonized broilers. In the second in vivo trial, the yolks were administered therapeutically during three days before euthanasia. The bacterin IgY resulted in a significant decrease in C. jejuni counts per infected bird. The hyperimmune yolks showed strong reactivity to a broad representation of C. jejuni and C. coli clonal complexes. These results indicate that passive immunization with hyperimmune yolks, especially bacterin derived, offers possibilities to control Campylobacter colonization in poultry

Probability to produce animal vaccines in insect baculovirus expression system

The insect baculovirus expression system is a valuable tool for the production of vaccine. Many subunit vaccines have been expressed in this system. The first vaccine produced in insect cells for animal use is now in the market. In this study, we reviewed recent progress of animal’s vaccine production for different expression levels and baculovirus genome stability, characteristic features of baculovirus expression vector system (BVES), virus link particles, baculovirus expression in mammalian cell and methodology of produce subunit vaccines. This review showed that BVES is a fantastic tool for vaccine development and it has wonderful feature for future animal vaccine development.Key words: Baculovirus expression system, vaccine, subunit vaccine, DNA vaccine

Acute retinal necrosis following recombinant subunit varicella-zoster virus vaccine.

PURPOSE: Previously, secondary prevention of herpes zoster required live-attenuated vaccination, which is contraindicated in immunocompromised populations. More recently, a recombinant subunit vaccine (Shingrix, GlaxoSmithKline, Research Triangle Park, North Carolina) was approved by the Food and Drug Administration. Iatrogenic varicella-zoster virus (VZV) infection is theoretically impossible as it does not contain a live virus. We present a case of acute retinal necrosis (ARN) and disseminated zoster after receiving the recombinant subunit vaccine. OBSERVATIONS: A 65-year-old woman with past medical history of multiple myeloma treated with a previous autologous hematopoietic stem cell transplant and now with daratumumab and pomalidomide developed disseminated zoster and subsequently acute retinal necrosis weeks after receiving the zoster subunit vaccine. Molecular testing confirmed the presence of VZV, and the absence of herpes simplex virus, cytomegalovirus, and toxoplasmosis. The VZV was found to be genotypically wildtype and not related to the Oka strain used in the live-attenuated zoster vaccine. She was treated with systemic valacyclovir and intravitreal foscarnet. CONCLUSIONS AND IMPORTANCE: This is the first report of VZV infection following the zoster subunit vaccine. The Advisory Committee on Immunization Practices (ACIP) has recommended the recombinant subunit vaccine over the live-attenuated vaccine due to its superior efficacy. The off-label use of the subunit vaccine in immunocompromised populations has been supported up to this point by studies demonstrating its relative safety. Though post-vaccination VZV infection or reactivation appears to be rare, clinicians should be aware of this potential complication to the recombinant subunit vaccine

Intranasal Delivery of Influenza Subunit Vaccine Formulated with GEM Particles as an Adjuvant

Nasal administration of influenza vaccine has the potential to facilitate influenza control and prevention. However, when administered intranasally (i.n.), commercially available inactivated vaccines only generate systemic and mucosal immune responses if strong adjuvants are used, which are often associated with safety problems. We describe the successful use of a safe adjuvant Gram-positive enhancer matrix (GEM) particles derived from the food-grade bacterium Lactococcus lactis for i.n. vaccination with subunit influenza vaccine in mice. It is shown that simple admixing of the vaccine with the GEM particles results in a strongly enhanced immune response. Already after one booster, the i.n. delivered GEM subunit vaccine resulted in hemagglutination inhibition titers in serum at a level equal to the conventional intramuscular (i.m.) route. Moreover, i.n. immunization with GEM subunit vaccine elicited superior mucosal and Th1 skewed immune responses compared to those induced by i.m. and i.n. administered subunit vaccine alone. In conclusion, GEM particles act as a potent adjuvant for i.n. influenza immunization

Production of a subunit vaccine candidate against porcine post-weaning diarrhea in high-biomass transplastomic tobacco

Post-weaning diarrhea (PWD) in piglets is a major problem in piggeries worldwide and results in severe economic losses. Infection with Enterotoxigenic Escherichia coli (ETEC) is the key culprit for the PWD disease. F4 fimbriae of ETEC are highly stable proteinaceous polymers, mainly composed of the major structural subunit FaeG, with a capacity to evoke mucosal immune responses, thus demonstrating a potential to act as an oral vaccine against ETEC-induced porcine PWD. In this study we used a transplastomic approach in tobacco to produce a recombinant variant of the FaeG protein, rFaeG(ntd/dsc), engineered for expression as a stable monomer by N-terminal deletion and donor strand-complementation (ntd/dsc). The generated transplastomic tobacco plants accumulated up to 2.0 g rFaeG(ntd/dsc) per 1 kg fresh leaf tissue (more than 1% of dry leaf tissue) and showed normal phenotype indistinguishable from wild type untransformed plants. We determined that chloroplast-produced rFaeG(ntd/dsc) protein retained the key properties of an oral vaccine, i.e. binding to porcine intestinal F4 receptors (F4R), and inhibition of the F4-possessing (F4+) ETEC attachment to F4R. Additionally, the plant biomass matrix was shown to delay degradation of the chloroplast-produced rFaeG(ntd/dsc) in gastrointestinal conditions, demonstrating a potential to function as a shelter-vehicle for vaccine delivery. These results suggest that transplastomic plants expressing the rFaeG(ntd/dsc) protein could be used for production and, possibly, delivery of an oral vaccine against porcine F4+ ETEC infections. Our findings therefore present a feasible approach for developing an oral vaccination strategy against porcine PWD

Computational modeling of TC0583 as a putative component of the Chlamydia muridarum V-type ATP synthase complex and assessment of its protective capabilities as a vaccine antigen.

Numerous Chlamydia trachomatis proteins have been identified as potential subunit vaccines, of which the major outer-membrane protein (MOMP) has, so far, proven the most efficacious. Recently, subunit A of the V-type ATP synthase (ATPase; TC0582) complex was shown to elicit partial protection against infection. Computational modeling of a neighboring gene revealed a novel subunit of the V-type ATPase (TC0583). To determine if this newly identified subunit could induce protection and/or enhance the partial protection provided by subunit A alone, challenge studies were performed using a combination of these recombinant proteins. The TC0583 subunit alone and concurrently with TC0582, was used to vaccinate BALB/c mice utilizing CpG-1826 and Montanide ISA 720 VG as adjuvants. Vaccinated animals were challenged intranasally with Chlamydia muridarum and the course of the infection was followed. Mice immunized with individual antigens showed minimal alleviation of body weight reduction; however, mice immunized with TC0583 and TC0582 in combination, displayed weight loss levels close to those observed with MOMP. Importantly, immunization with a combination of recombinant subunit proteins reduced chlamydial inclusion forming units by approximately a log-fold. These protection levels support that, these highly conserved Chlamydia proteins, in combination with other antigens, may serve as potential vaccine candidates