Development of a Fully Protective Pandemic Avian Influenza Subunit Vaccine in Insect Pupae.

In this study, we pioneered an alternative technology for manufacturing subunit influenza hemagglutinin (HA)-based vaccines. This innovative method involves harnessing the pupae of the Lepidoptera Trichoplusia ni (T. ni) as natural biofactories in combination with baculovirus vectors (using CrisBio® technology). We engineered recombinant baculoviruses encoding two versions of the HA protein (trimeric or monomeric) derived from a pandemic avian H7N1 virus A strain (A/chicken/Italy/5093/99). These were then used to infect T. ni pupae, resulting in the production of the desired recombinant antigens. The obtained HA proteins were purified using affinity chromatography, consistently yielding approximately 75 mg/L of insect extract. The vaccine antigen effectively immunized poultry, which were subsequently challenged with a virulent H7N1 avian influenza virus. Following infection, all vaccinated animals survived without displaying any clinical symptoms, while none of the mock-vaccinated control animals survived. The CrisBio®-derived antigens induced high titers of HA-specific antibodies in the vaccinated poultry, demonstrating hemagglutination inhibition activity against avian H7N1 and human H7N9 viruses. These results suggest that the CrisBio® technology platform has the potential to address major industry challenges associated with producing recombinant influenza subunit vaccines, such as enhancing production yields, scalability, and the speed of development, facilitating the global deployment of highly effective influenza vaccines.

Enhanced Recombinant Protein Expression in Insect Cells by Natural and Recombinant Components of Lepidoptera Hemolymph.

Prior research has established the anti-apoptotic effects in insect cell cultures of Bombyx mori (B. mori) hemolymph, as well as the heightened production yields of recombinant proteins facilitated by baculovirus vectors in insect cells cultivated in media supplemented with this hemolymph. In this study, we investigated the hemolymph of another Lepidoptera species, Trichoplusia ni (T. ni), and observed similar beneficial effects in insect cells cultivated in media supplemented with this natural substance. We observed enhancements in both production yield (approximately 1.5 times higher) and late-stage cell viabilities post-infection (30-40% higher). Storage-protein 2 from B. mori (SP2Bm) has previously been identified as one of the abundant hemolymph proteins potentially responsible for the beneficial effects observed after the use of B. mori hemolymph-supplemented cell culture media. By employing a dual baculovirus vector that co-expresses the SP2Bm protein alongside the GFP protein, we achieved a threefold increase in reporter protein production compared to a baculovirus vector expressing GFP alone. This study underscores the potential of hemolymph proteins sourced from various Lepidoptera species as biotechnological tools to augment baculovirus vector productivities, whether utilized as natural supplements in cell culture media or as hemolymph-derived recombinant proteins co-expressed by baculovirus vectors.

Chrysalises as natural production units for recombinant subunit vaccines.

The baculovirus vector expression system (BEVS) combines cultured insect cells and genetically modified Autographa californica nuclear polyhedrosis virus (AcMNPV)-derived baculovirus vectors. This expression system has been widely used for the expression of hundred of proteins for more than 30 years, existing commercial products manufactured at large scale by this methodology, mainly subunit vaccines. At an industrial scale, insect cells, as any other cultured cells, require artificial media and a strict control of environmental sterile conditions in the complex and expensive bioreactors. Here we describe an efficient alternative to produce recombinant biologics using the versatile and productive baculovirus vectors. It consists in natural biocapsules (pupae from Trichoplusia ni (Hübner) Lepidoptera), containing millions of insect cells in perfect physiological conditions, ready to be programmed by a genetically modified AcMNPV-derived baculovirus vector to produce large quantities of any recombinant protein. This technology, denominated CrisBio, has been tested to produce dozens of proteins, reaching productivities on the range of milligrams per infected pupa, that can be translated into dozens of vaccine doses, for example. The biologics production by CrisBio was industrialized with the design of both insect rearing and pupae storage single-use plastic devices, compatible with machines specifically designed for the automation of pupae manipulation and inoculation. These devices and machines reduce manual operations, increase batches consistency and facilitate the scaled production of any recombinant protein. As a mode of examples, the productivity in CrisBio technology platform of two virus-like particle (VLP) vaccine antigens is described in this work.

Conserved HA-peptides expressed along with flagellin in Trichoplusia ni larvae protects chicken against intranasal H7N1 HPAIV challenge.

The immunization of poultry where H5 and H7 influenza viruses (IVs) are endemic is one of the strategies to prevent unexpected zoonoses. Our group has been focused on conserved HA-epitopes as potential vaccine candidates to obtain multivalent immune responses against distinct IV subtypes. In this study, two conserved epitopes (NG-34 and CS-17) fused to flagellin were produced in a Baculovirus platform based on Trichoplusia ni larvae as living biofactories. Soluble extracts obtained from larvae expressing "flagellin-NG34/CS17 antigen" were used to immunize chickens and the efficacy of the vaccine was evaluated against a heterologous H7N1 HPAIV challenge in chickens. The flagellin-NG34/CS17 vaccine protected the vaccinated chickens and blocked viral shedding orally and cloacally. Furthermore, no apparent clinical signs were monitored in 10/12 vaccinated individuals. The mechanism of protection conferred is under investigation.

Rational design of protamine nanocapsules as antigen delivery carriers.

Current challenges in global immunization indicate the demand for new delivery strategies, which could be applied to the development of new vaccines against emerging diseases, as well as to improve safety and efficacy of currently existing vaccine formulations. Here, we report a novel antigen nanocarrier consisting of an oily core and a protamine shell, further stabilized with pegylated surfactants. These nanocarriers, named protamine nanocapsules, were rationally designed to promote the intracellular delivery of antigens to immunocompetent cells and to trigger an efficient and long-lasting immune response. Protamine nanocapsules have nanometric size, positive zeta potential and high association capacity for H1N1 influenza hemagglutinin, a protein that was used here as a model antigen. The new formulation shows an attractive stability profile both, as an aqueous suspension or a freeze-dried powder formulation. In vitro studies showed that protamine nanocapsules were efficiently internalized by macrophages without eliciting significant toxicity. In vivo studies indicate that antigen-loaded nanocapsules trigger immune responses comparable to those achieved with alum, even when using significantly lower antigen doses, thus indicating their adjuvant properties. These promising in vivo data, alongside with their versatility for the loading of different antigens and oily immunomodulators and their excellent stability profile, make these nanocapsules a promising platform for the delivery of antigens. CHEMICAL COMPOUNDS: Protamine sulphate (PubChem SID: 7849283), Sodium Cholate (PubChem CID: 23668194), Miglyol (PubChem CID: 53471835), α tocopherol (PubChem CID: 14985), Tween® 20(PubChem CID: 443314), Tween® 80(PubChem CID: 5281955), TPGS (PubChem CID: 71406).

Highly versatile immunostimulating nanocapsules for specific immune potentiation.

AIM: To develop a new core-shell type (nanocapsules) adjuvant system composed of squalene and polyglucosamine (PG) and to evaluate its immunostimulant capacity. RESULTS: The defined PG nanocapsules exhibited the capacity to efficiently associate the selected antigens (recombinant hepatitis B surface antigen and hemagglutinin of influenza virus) onto their polymeric surface (70-75%), and the immunostimulant imiquimod within the oily core. The resulting nanovaccines, with a particle size of 200-250 nm and a positive zeta-potential (∼+60 mV), were able to significantly potentiate and modulate the immune response to the selected antigens upon intramuscular administration to mice. Their efficacy as novel adjuvants was attributed to their enhanced cell internalization and effective intracellular imiquimod/antigen delivery, together with their prolonged residence time at the injection site. CONCLUSION: The nanocapsules described herein have the capacity to enhance, prolong and modulate the immune response of subunit antigens and, therefore, they could be proposed as a platform for the codelivery of different antigens and immunostimulators.

Kinetics and role of antibodies against intimin beta in colostrum and in serum from goat kids and longitudinal study of attaching and effacing Escherichia coli in goat kids.

The presence of antibodies to the intimin beta-binding region (Int280-beta) of attaching and effacing Escherichia coli (AEEC) in serum from 20 goat kids from 2 herds, as well as in goat colostrum, was investigated by enzyme-linked immunosorbent assay. In addition, the onset and subsequent pattern of shedding of AEEC from the same goat kids over a 6-mo period was investigated. All the colostrum and serum samples tested contained antibodies against Int280-beta. The association between the antibody titer and the isolation of AEEC suggests that antibodies to intimin beta do not prevent colonization of the intestine by AEEC in goat kids. The AEEC were generally shed only transiently. Most AEEC isolated from the kids belonged to serogroup O26. Three isolates belonged to serogroup O157. These data show that goat kids may be a reservoir of AEEC that are potentially pathogenic for humans.

Restoring catalase activity in Staphylococcus aureus subsp. anaerobius leads to loss of pathogenicity for lambs.

Staphylococcus aureus subsp. anaerobius, a microaerophilic and catalase-negative bacterium, is the etiological agent of abscess disease, a specific chronic condition of sheep and goats, which is characterized by formation of necrotic lesions that are located typically in superficial lymph nodes. We constructed an isogenic mutant of S. aureus subsp. anaerobius (RDKA84) that carried a repaired and functional catalase gene from S. aureus ATCC 12600, to investigate whether the lack of catalase in S. aureus subsp. anaerobius plays a role in its physiological and pathogenic characteristics. The catalase activity had no apparent influence on the in vitro growth characteristics of RDKA84, which, like the wild-type, did not grow on aerobically incubated agar plates. Restoration of catalase activity in RDKA84 substantially increased resistance to H2O2 when analyzed in a death assay. The intracellular survival rates of the catalase-positive mutant RDKA84 in polymorphonuclear neutrophils (PMN) isolated from adult sheep were significantly higher than those of the wild-type, while no differences were found with PMN isolated from lambs. RDKA84 showed significantly lower survival rates in murine macrophages (J774A.1 cells) than the wild-type strains did, whereas, in bovine mammary epithelial cells (MAC-T), no differences in intracellular survival were observed. Interestingly, the virulence for lambs, the natural host for abscess disease, of the catalase-positive mutant RDKA84 was reduced dramatically in comparison with wild-type S. aureus subsp. anaerobius in two experimental models of infection.

Simultaneous lack of catalase and beta-toxin in Staphylococcus aureus leads to increased intracellular survival in macrophages and epithelial cells and to attenuated virulence in murine and ovine models.

Staphylococcus aureus produces a variety of virulence factors that allow it to cause a wide range of infections in humans and animals. In the latter, S. aureus is a leading cause of intramammary infections. The contribution of catalase (KatA), an enzyme implicated in oxidative stress resistance, and beta-toxin (Hlb), a haemolysin, to the pathogenesis of S. aureus is poorly characterized. To investigate the role of these enzymes as potential virulence factors in S. aureus, we examined the intracellular survival of DeltakatA, Deltahlb and DeltakatA Deltahlb mutants in murine macrophages (J774A.1) and bovine mammary epithelial cells (MAC-T), and their virulence in different murine and ovine models. Catalase was not required for the survival of S. aureus within either J774A.1 or MAC-T cells. However, it was necessary for the intracellular proliferation of the bacterium after invasion of MAC-T cells. In addition, catalase was not needed for the full virulence of S. aureus in mice. Deletion of the hlb gene had no effect on the intracellular survival of S. aureus in J774A.1 cells but did cause a slight increase in survival in MAC-T cells. Furthermore, like catalase, beta-toxin was not required for complete virulence of S. aureus in murine models. Unexpectedly, the DeltakatA Deltahlb mutant showed a notably increased persistence in both cell lines, and was significantly less virulent for mice than were the wild-type strain and single mutants. Most interestingly, it was also markedly attenuated in intramammary and subcutaneous infections in ewes and lambs.

Polymerase chain reaction typing of genes of the locus of enterocyte effacement of ruminant attaching and effacing Escherichia coil.

The variability of the tir, espA, and espD genes of the locus of enterocyte effacement (LEE) in 185 attaching and effacing Escherichia coli (AEEC) strains isolated from healthy and diarrheic cattle, sheep, and goats was investigated by polymerase chain reaction. Nineteen of the strains were enterohemorrhagic E. coli (EHEC); the other 166 were enteropathogenic E. coli (EPEC). The combinations of the tir and esp genes were associated with the variants of the eae gene but not with a strain's belonging to the EPEC or EHEC group, animal species, or health status (healthy or diarrheic) of the animal. In addition, most of the strains showed the same combinations of LEE genes and serogroups as have been found in AEEC strains isolated from humans, which indicates that ruminants seem to be an EPEC reservoir for humans.

Characterization of fluoroquinolone resistance in Escherichia coli strains from ruminants.

Thirty-seven fluoroquinolone-resistant Escherichia coli strains from ruminants (according to Clinical and Laboratory Standards Institute guidelines) were screened by molecular methods for mutations in the quinolone resistance-determining region (QRDR) of the gyrA and parC genes and for the presence of the qnrA gene. One of the strains studied was an enterohemorrhagic E. coli (EHEC) strain potentially pathogenic for humans. Three E. coli strains resistant to enrofloxacin (minimal inhibitory concentration [MIC] = 2 microg/ml) but not to ciprofloxacin (MIC = 1 microg/ml) presented single mutations in the gyrA and parC genes, while 34 strains resistant to both fluoroquinolones presented double and single mutations in gyrA and parC, respectively (31 strains), or double mutations in gyrA and parC (3 strains). The EHEC strain presented a double amino acid substitution in the GyrA protein (Ser-83-->Leu and Asp-87-->Gly) and a double amino acid substitution in the ParC protein (Gly-78-->Cys and Ser-80-->Arg), one of which has not been previously described. The present study shows that most of the mutations in the QRDR of the gyrA and parC genes of fluoroquinolone-resistant E. coli strains from ruminants are the same as those seen in E. coli strains from other animal species and humans and that there are no differences in mutation patterns in the QRDR of E. coli strains from healthy ruminants and those with diarrhea. No strains carried qnrA, which indicates that this gene does not play an important role in the selection of fluoroquinolone-resistant E. coli strains from ruminants.

Salmonella enterica serovar Choleraesuis derivatives harbouring deletions in rpoS and phoP regulatory genes are attenuated in pigs, and survive and multiply in porcine intestinal macrophages and fibroblasts, respectively.

Live attenuated Salmonella enterica strains have been extensively studied as potential vectors for the oral delivery of heterologous antigens. Due to its ability to target immune cells, its specific mechanism for crossing the intestinal barrier, and its swine-restricted tropism, S. enterica subspecies enterica serovar Choleraesuis (S. Choleraesuis) has attracted a great deal of interest for the production of bacterial-based oral carriers specifically adapted to swine. In this study, two mutants of S. Choleraesuis were constructed and their attenuation and intracellular fate analysed with the purpose of engineering new attenuated live strains with improved properties as oral vaccine carriers. Those strains harboured a specific deletion either within the phoP or rpoS genes, which encode virulence-related regulators in S. Typhimurium. In comparison to the wild-type parental S. Choleraesuis, the mutant strains, especially DeltaphoP, were extremely low in virulence in the murine model and in the natural host, the pig. Moreover, when compared with a commercial live vaccine strain, SC-54, the two mutants showed a higher level of attenuation in mice and DeltaphoP also in pigs. In addition, DeltarpoS and DeltaphoP presented a proliferation and survival phenotype within swine intestinal primary fibroblast and macrophage cell cultures, respectively. Collectively, the present results indicate that the DeltarpoS and DeltaphoP strains of S. Choleraesuis gather adequate features to be potential candidates for vaccine vectors for the specific delivery of heterologous antigens adapted to pigs.

Necrotoxigenic Escherichia coli from sheep and goats produce a new type of cytotoxic necrotizing factor (CNF3) associated with the eae and ehxA genes.

Fecal samples from sheep and goats were screened by tissue-culture assays and PCR for the presence of necrotoxigenic Escherichia coli (NTEC) producing cytotoxic necrotizing factors (CNFs). Of the 18 NTEC strains assayed, four were positive for the cnf1 gene while 14 strains were negative for the cnf1 and cnf2 genes. All of the NTEC strains had the eae gene and most of them also carried the ehxA gene. Moreover, all the cnf1- cnf2- NTEC strains were negative for several virulence markers associated with CNF1+ or CNF2+ strains. The cnf gene present in one of these strains was sequenced and analysis of the gene product revealed a new type of CNF, which was named CNF3 (and the coding gene cnf3). Oligonucleotide primers were designed to PCR-amplify a fragment of cnf3. The results showed that all strains examined in this study, except one cnf1+strain, were cnf3+. The association of cnf3 with eae and ehxA suggests that cnf3+ NTEC strains might be pathogenic for humans.

Necrotoxigenic Escherichia coli from sheep and goats produce a new type of cytotoxic necrotizing factor (CNF3) associated with the eae and ehxA genes

Fecal samples from sheep and goats were screened by tissue-culture assays and PCR for the presence of necrotoxigenic Escherichia coli (NTEC) producing cytotoxic necrotizing factors (CNFs). Of the 18 NTEC strains assayed, four were positive for the cnf1 gene while 14 strains were negative for the cnf1 and cnf2 genes. All of the NTEC strains had the eae gene and most of them also carried the ehxA gene. Moreover, all the cnf1- cnf2- NTEC strains were negative for several virulence markers associated with CNF1+ or CNF2+ strains. The cnf gene present in one of these strains was sequenced and analysis of the gene product revealed a new type of CNF, which was named CNF3 (and the coding gene cnf3). Oligonucleotide primers were designed to PCR-amplify a fragment of cnf3. The results showed that all strains examined in this study, except one cnf1+strain, were cnf3+. The association of cnf3 with eae and ehxA suggests that cnf3+ NTEC strains might be pathogenic for humans (AU)

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