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Drug-resistant tuberculosis (DR-TB) poses a new threat to global health; to improve the treatment outcome, therapeutic vaccines are considered the best chemotherapy adjuvants. Unfortunately, there is no therapeutic vaccine approved against DR-TB. Our study assessed the therapeutic efficacy of a recombinant drug-resistant BCG (RdrBCG) vaccine in DR-TB. We constructed the RdrBCG overexpressing Ag85B and Rv2628 by selecting drug-resistant BCG strains and transformed them with plasmid pEBCG or pIBCG to create RdrBCG-E and RdrBCG-I respectively. Following successful stability testing, we tested the vaccine's safety in severe combined immune deficient (SCID) mice that lack both T and B lymphocytes plus immunoglobulins. Finally, we evaluated the RdrBCG's therapeutic efficacy in BALB/c mice infected with rifampin-resistant M. tuberculosis and treated with a second-line anti-TB regimen. We obtained M. bovis strains which were resistant to several second-line drugs and M. tuberculosis resistant to rifampin. Notably, the exogenously inserted genes were lost in RdrBCG-E but remained stable in the RdrBCG-I both in vitro and in vivo. When administered adjunct to a second-line anti-TB regimen in a murine model of DR-TB, the RdrBCG-I lowered lung M. tuberculosis burden by 1 log 10 . Furthermore, vaccination with RdrBCG-I adjunct to chemotherapy minimized lung tissue pathology in mice. Most importantly, the RdrBCG-I showed almost the same virulence as its parent BCG Tice strain in SCID mice. Our findings suggested that the RdrBCG-I was stable, safe and effective as a therapeutic vaccine. Hence, the "recombinant" plus "drug-resistant" BCG strategy could be a useful concept for developing therapeutic vaccines against DR-TB., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Since the successful use of vaccinia virus (VACV) in the immunization strategies to eliminate smallpox, research has been focused on the development of recombinant VACV strains expressing proteins from various pathogens. Attempts at decreasing the side effects associated with exposure to recombinant, wild-type viral strains have led to the development of attenuated viruses. Yet while these attenuated VACV's have improved safety profiles compared to unmodified strains, their clinical use has been hindered due to efficacy issues in stimulating a host immune response. This deficiency has largely been attributed to decreased production of the target protein for immunization. Efforts to increase protein production from attenuated VACV strains has largely centered around modulation of viral factors, while manipulation of the translation of viral mRNAs has been largely unexplored. In this study we evaluate the use of translation enhancing element hTEE-658 to increase recombinant protein production in an attenuated VACV system. Optimization of the use of this motif is also attempted by combining it with strategies that have demonstrated effectiveness in previous research. We show that extension of the 5' leader sequence containing hTEE-658 does not improve motif function, nor does the combination with other known translation enhancing elements. However, the sole use of hTEE-658 in an attenuated VACV system is shown to increase protein expression levels beyond those of a standard viral promoter when used with a wild-type virus. Taken together these results highlight the potential for hTEE-658 to improve the effectiveness of attenuated VACV vaccine candidates and give insights into the optimal sequence context for its use in vaccine design.

A self-transcribing and replicating RNA (STARR)-based vaccine (LUNAR-COV19) has been developed to prevent SARS-CoV-2 infection. The vaccine encodes an alphavirus-based replicon and the SARS-CoV-2 full-length spike glycoprotein. Translation of the replicon produces a replicase complex that amplifies and prolongs SARS-CoV-2 spike glycoprotein expression. A single prime vaccination in mice led to robust antibody responses, with neutralizing antibody titers increasing up to day 60. Activation of cell-mediated immunity produced a strong viral antigen-specific CD8 + T lymphocyte response. Assaying for intracellular cytokine staining for interferon (IFN)γ and interleukin-4 (IL-4)-positive CD4 + T helper (Th) lymphocytes as well as anti-spike glycoprotein immunoglobulin G (IgG)2a/IgG1 ratios supported a strong Th1-dominant immune response. Finally, single LUNAR-COV19 vaccination at both 2 μg and 10 μg doses completely protected human ACE2 transgenic mice from both mortality and even measurable infection following wild-type SARS-CoV-2 challenge. Our findings collectively suggest the potential of LUNAR-COV19 as a single-dose vaccine., Competing Interests: Declaration of interests D.M., E.C.A., P.H., K.-J.J.P., M.A., H.B., A.D., Y.B., B.C., J.V., S.R, J.A.G., M.S., R.Y., W.T., K.T., S.P., P.K., J.D., S.M.S., B.M.S, S.G.H., and P.C. are employees of Arcturus Therapeutics, Inc. E.E.O is an unpaid member of the Scientific Advisory Board of Arcturus Therapeutics, Inc., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

A key lesson emerging from COVID-19 is that pandemic proofing planetary health against future ecological crises calls for systems science and preventive medicine innovations. With greater proximity of the human and animal natural habitats in the 21st century, it is also noteworthy that zoonotic infections such as COVID-19 that jump from animals to humans are increasingly plausible in the coming decades. In this context, glycomics technologies and the third alphabet of life, the sugar code, offer veritable prospects to move omics systems science from discovery to diverse applications of relevance to global public health and preventive medicine. In this expert review, we discuss the science of glycomics, its importance in vaccine development, and the recent progress toward discoveries on the sugar code that can help prevent future infectious outbreaks that are looming on the horizon in the 21st century. Glycomics offers veritable prospects to boost planetary health, not to mention the global scientific capacity for vaccine innovation against novel and existing infectious agents.

Self-amplifying RNA (saRNA) is a cutting-edge platform for both nucleic acid vaccines and therapeutics. saRNA is self-adjuvanting, as it activates types I and III interferon (IFN), which enhances the immunogenicity of RNA vaccines but can also lead to inhibition of translation. In this study, we screened a library of saRNA constructs with cis-encoded innate inhibiting proteins (IIPs) and determined the effect on protein expression and immunogenicity. We observed that the PIV-5 V and Middle East respiratory syndrome coronavirus (MERS-CoV) ORF4a proteins enhance protein expression 100- to 500-fold in vitro in IFN-competent HeLa and MRC5 cells. We found that the MERS-CoV ORF4a protein partially abates dose nonlinearity in vivo, and that ruxolitinib, a potent Janus kinase (JAK)/signal transducer and activator of transcription (STAT) inhibitor, but not the IIPs, enhances protein expression of saRNA in vivo. Both the PIV-5 V and MERS-CoV ORF4a proteins were found to enhance the percentage of resident cells in human skin explants expressing saRNA and completely rescued dose nonlinearity of saRNA. Finally, we observed that the MERS-CoV ORF4a increased the rabies virus (RABV)-specific immunoglobulin G (IgG) titer and neutralization half-maximal inhibitory concentration (IC 50 ) by ∼10-fold in rabbits, but not in mice or rats. These experiments provide a proof of concept that IIPs can be directly encoded into saRNA vectors and effectively abate the nonlinear dose dependency and enhance immunogenicity., Competing Interests: Declaration of Interests A.K.B., P.F.M., and R.J.S. are co-inventors of a patent resulting from this work. The remaining authors declare no competing interests., (Crown Copyright © 2020. Published by Elsevier Inc. All rights reserved.)

Coronaviruses (CoVs) are nonsegmented, single-stranded, positive-sense RNA viruses highly pathogenic to humans. Some CoVs are known to cause respiratory and intestinal diseases, posing a threat to the global public health. Against this backdrop, it is of critical importance to develop safe and effective vaccines against these CoVs. This review discusses human vaccine candidates in any stage of development and explores the viral characteristics, molecular epidemiology, and immunology associated with CoV vaccine development. At present, there are many obstacles and challenges to vaccine research and development, including the lack of knowledge about virus transmission, pathogenesis, and immune response, absence of the most appropriate animal models., (© 2020 Wiley Periodicals LLC.)

The cysteine-rich Pfs48/45 protein, a Plasmodium falciparum sexual stage surface protein, has been advancing as a candidate antigen for a transmission-blocking vaccine (TBV) for malaria. However, Pfs48/45 contains multiple disulfide bonds, that are critical for proper folding and induction of transmission-blocking (TB) antibodies. We have previously shown that R0.6C, a fusion of the 6C domain of Pfs48/45 and a fragment of PfGLURP (R0), expressed in Lactococcus lactis , was properly folded and induced transmission-blocking antibodies. Here we describe the process development and technology transfer of a scalable and reproducible process suitable for R0.6C manufacturing under current Good Manufacturing Practices (cGMP). This process resulted in a final purified yield of 25 mg/L, sufficient for clinical evaluation. A panel of analytical assays for release and stability assessment of R0.6C were developed including HPLC, SDS-PAGE, and immunoblotting with the conformation-dependent TB mAb45.1. Intact mass analysis of R0.6C confirmed the identity of the product including the three disulfide bonds and the absence of post-translational modifications. Multi-Angle Light Scattering (MALS) coupled to size exclusion chromatography (SEC-MALS), further confirmed that R0.6C was monomeric (~70 kDa) in solution. Lastly, preclinical studies demonstrated that the R0.6C Drug Product (adsorbed to Alhydrogel ® ) elicited functional antibodies in small rodents and that adding Matrix-M ™ adjuvant further increased the functional response. Here, building upon our past work, we filled the gap between laboratory and manufacturing to ready R0.6C for production under cGMP and eventual clinical evaluation as a malaria TB vaccine., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Singh, Plieskatt, Chourasia, Fabra-García, Garcia-Senosiain, Singh, Bengtsson, Reimer, Sauerwein, Jore and Theisen.)

Background: Leishmaniasis is a serious health problem in some parts of the world. In spite of the many known leishmaniasis control measures, the disease has continued to increase in endemic areas, and no effective vaccine has been discovered., Methods: In this study, Leishmania tarentulae was used as a living factory for the production of two LACK and KMP11 immunogenic antigens in the mice body, and safety profiles were investigated. The sequences of the KMP11 and LACK L. major antigens were synthesized in the pLEXSY-neo 2.1 plasmid and cloned into E. coli strain Top10, and after being linearized with the SwaI enzyme, they were transfected into the genome of L. tarentolae. The L. tarentolae-LACK/KMP11/EGFP in the stationary phase with CpG ODN as an adjuvant was used for vaccination in BALB/c mice. Vaccination was performed into the left footpad. Three weeks later, the booster was injected in the same manner. To examine the effectiveness of the injected vaccine, pathogenic L. major (MRHO/IR/75/ER) was injected into the right footpad of all mice three weeks following the booster vaccination. In order to assess humoral immunity, the levels of IgG1, and IgG2a antibodies before and 6 weeks after the challenge were studied in the groups. In addition, in order to investigate cellular immunity in the groups, the study measured IFN-γ, IL-5, TNF-α, IL-6 and IL-17 cytokines before, 3 weeks and 8 weeks after the challenge, and also the parasite load in the lymph node with real-time PCR., Results: The lowest level of the parasitic load was observed in the G1 group (mice vaccinated with L. tarentolae-LACK/KMP11/EGFP with CpG) in comparison with other groups (L. tarentolae-LACK/KMP11/EGFP +non-CpG (G2); L. tarentolae-EGFP + CpG (G3, control); L. tarentolae-EGFP + non-CpG (G4, control); and mice injected with PBS (G5, control). Moreover, the evaluation of immune response showed a delayed-type hypersensitivity towards Th1., Conclusions: According to the results of this study, the live recombinant vaccine of L. tarentolae-LACK/KMP11/EGFP with the CpG adjuvant reduced the parasitic load and footpad induration in infected mice. The long-term effects of this vaccine can be evaluated in volunteers as a clinical trial in future planning.

Escherichia coli O157:H7 and Shigella flexneri are the predominant diarrhoeal pathogens and those strains producing Shiga toxins cause life-threatening sequelae including hemolytic uremic syndrome (HUS) upon their entry into the host. Intimate adherence of E. coli O157 and invasion of S. flexneri in the host intestinal epithelial cells is mainly mediated by Intimin and IpaB proteins, respectively. In this study, we have synthesized chimera of immunodominant regions of Intimin (eae) and IpaB (ipaB) designated as EI and expressed it in Lactococcus lactis (LL-EI) to develop a combinatorial oral vaccine candidate. Immune parameters and protective efficacy of orally administered LL-EI were assessed in the murine model. Significant EI-specific serum IgG, IgA, and fecal IgA antibody titer were observed in the LL-EI group. Considerable increase in EI-specific splenocyte proliferation and a concurrent upregulation of both Th1 and Th2 cytokines was observed in LL-EI immunized mice. Flow cytometry analysis also revealed a significant increase in CD4 and CD8 cell counts in LL-EI immunized group compared to PBS, LL control group.In vitro studies using LL-EI immunized mice sera showed substantial protection against bacterial adhesion and invasion caused by E. coli O157 and Shigella flexneri¸ respectively. LL-EI immunized group challenged with E. coli O157 ceased fecal shedding within 6 days, and mice challenged with S. flexneri showed 93% survival with minimal bacterial load in the lungs. Our results indicate that LL-EI immunization elicits systemic, mucosal and cell-mediated immune responses, and can be a promising candidate for oral vaccine development against these pathogens., Competing Interests: Declaration of Competing Interest Authors authenticate no conflict of interest among them., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Ebola virus disease outbreaks have repeatedly occurred on the African continent over the last decades, with more serious outbreaks in recent years. Being highly transmissible and associated to high fatality rates, it constitutes a serious threat to public health. Vaccination, however, may allow for efficient control of its propagation. The most promising Ebola vaccine candidate to date, rVSV-ZEBOV, relies on a recombinant vesicular stomatitis virus construct, in which the native viral glycoprotein is replaced by the glycoprotein of Ebola virus (Zaire). However, its cell-based manufacturing process is still lengthy and cumbersome, thus urging the implementation of a new and more efficient bioprocess. To address these issues, serum-free production of rVSV-ZEBOV in Vero cells has been studied with the aim to test an alternative upstream process. Until viable options of suspension cell culture are available, Vero cell cultures still rely on adherent bioprocesses and have thus been developed in this work. Particularly, a bioprocess developed with standard microcarrier bioreactor technology was successfully transferred to the novel single-use scale-X™ hydro fixed-bed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Recombinant proteins expressed in host cell systems may contain host cell proteins (HCP) as impurities. While there is no clear evidence of clinical adverse events attributable to HCP, HCP levels and profiles must be documented to meet regulatory requirements and to understand the consistency of the biological product and manufacturing process. We present a general strategy for HCP characterization applied to a recombinant protein antigen, Hepatitis B surface antigen (HBsAg) used in a multivalent vaccine., Methods: Polyclonal antisera raised against HCPs in process fractions from a mock preparation of the HBsAg yeast expression host, Hansenula polymorpha, were used to develop a quantitative sandwich ELISA to measure HCP content in batches of purified recombinant HBsAg. Batches were also subjected to SDS-PAGE and LC-MS/MS to identify detectable proteins. Batch consistency was further assessed by SDS-PAGE/densitometry purity analysis and by the ratio of specific HBsAg content (by ELISA) to total protein., Results: Using the quantitative HCP ELISA, the HCP content showed no discernable trend in multiple HBsAg batches manufactured over a 5-year period. All batches were ≥95% pure by SDS-PAGE/densitometry, with consistent HBsAg/total protein ratios. In addition to the expected HBsAg antigen protein, LC-MS/MS analysis of three HBsAg batches identified several yeast proteins, none of which are known to cause adverse reactions in humans., Conclusions: Analysis of multiple HBsAg batches showed consistent HCP content and identification profiles, as well as product purity and specific antigen content, demonstrating consistent manufacturing process. Recombinant vaccines, unlike therapeutic products, are administered infrequently with only small amounts of protein injected at a time. With limited potential for adverse reactions to small quantities of HCPs in purified recombinant vaccine antigens, and considering the relevant regulatory guidelines, we conclude that once consistent manufacturing process has been demonstrated, routine HCP testing in recombinant vaccine antigens is no longer required., (Copyright © 2018. Published by Elsevier Ltd.)

Main Conclusion: We demonstrated successful overexpression of porcine reproductive and respiratory syndrome virus (PRRSV)-derived GP4D and GP5D antigenic proteins in Arabidopsis. Pigs immunized with transgenic plants expressing GP4D and GP5D proteins generated both humoral and cellular immune responses to PRRSV. Porcine reproductive and respiratory syndrome virus (PRRSV) causes PRRS, the most economically significant disease affecting the swine industry worldwide. However, current commercial PRRSV vaccines (killed virus or modified live vaccines) show poor efficacy and safety due to concerns such as reversion of virus to wild type and lack of cross protection. To overcome these problems, plants are considered a promising alternative to conventional platforms and as a vehicle for large-scale production of recombinant proteins. Here, we demonstrate successful production of recombinant protein vaccine by expressing codon-optimized and transmembrane-deleted recombinant glycoproteins (GP4D and GP5D) from PRRSV in planta. We generated transgenic Arabidopsis plants expressing GP4D and GP5D proteins as candidate antigens. To examine immunogenicity, pigs were fed transgenic Arabidopsis leaves expressing the GP4D and GP5D antigens (three times at 2-week intervals) and then challenged with PRRSV at 6-week post-initial treatment. Immunized pigs showed significantly lower lung lesion scores and reduced viremia and viral loads in the lung than pigs fed Arabidopsis leaves expressing mYFP (control). Immunized pigs also had higher titers of PRRSV-specific antibodies and significantly higher levels of pro-inflammatory cytokines (TNF-α and IL-12). Furthermore, the numbers of IFN-γ + -producing cells were higher, and those of regulatory T cells were lower, in GP4D and GP5D immunized pigs than in control pigs. Thus, plant-derived GP4D and GP5D proteins provide an alternative platform for producing an effective subunit vaccine against PRRSV.

Background: Influenza is a widely distributed infection that almost annually causes seasonal epidemics. The current egg-based platforms for influenza vaccine production are facing a number of challenges and are failing to satisfy the global demand in the case of pandemics due to the long production time. Recombinant vaccines are an alternative that can be quickly produced in high quantities in standard expression systems., Methods: Plants may become a promising biofactory for the large-scale production of recombinant proteins due to low cost, scalability, and safety. Plant-based expression systems have been used to produce recombinant vaccines against influenza based on two targets; the major surface antigen hemagglutinin and the transmembrane protein M2., Results: Different forms of recombinant hemagglutinin were successfully expressed in plants, and some plantproduced vaccines based on hemagglutinin were successfully tested in clinical trials. However, these vaccines remain strain specific, while the highly conserved extracellular domain of the M2 protein (M2e) could be used for the development of a universal influenza vaccine. In this review, the state of the art in developing plant-produced influenza vaccines based on M2e is presented and placed in perspective. A number of strategies to produce M2e in an immunogenic form in plants have been reported, including its presentation on the surface of plant viruses or virus-like particles formed by capsid proteins, linkage to bacterial flagellin, and targeting to protein bodies., Conclusion: Some M2e-based vaccine candidates were produced at high levels (up to 1 mg/g of fresh plant tissue) and were shown to be capable of stimulating broad-range protective immunity., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Newcastle disease virus (NDV) recombinants expressing the infectious laryngotracheitis virus (ILTV) glycoproteins B and D have previously been demonstrated to confer complete clinical protection against virulent ILTV and NDV challenges in naive chickens. We extended this study to assess whether maternally derived antibody (MDA) against NDV and ILTV would interfere with protection in vaccinated broiler chickens. Chickens with a mean NDV MDA hemagglutination inhibition (HI) titer of 6.4 (log 2 ) and detectable ILTV neutralization (VN) antibodies at hatch were vaccinated with rLS/ILTV-gB or rLS/ILTV-gD at 1 or 10day of age (DOA) or on both days. Groups of birds vaccinated with the commercial ILT vaccines (FP-LT and CEO) or sham inoculated were also included in this study. All vaccinated birds were challenged with virulent ILTV strain at 21 DOA. By that time, NDV HI titers declined to 2.6 (log 2 ) in unvaccinated birds, whereas the HI titers in NDV vectored vaccine groups increased to 3.5-6.3 (log 2 ). At standard dosages, both vaccine candidates conferred significant clinical protection; however, the protection elicited by the rLS/ILTV-gD was superior to that of rLS/ILTV-gB. Recombinant rLS/ILTV-gD reduced ILTV shedding from tracheal and ocular tissues by approximately 3 log 10 TCID 50 . Notably, there was no improvement in protection after booster vaccination at 10 DOA. Overall results indicate that the presence of maternal antibodies to NDV and ILTV did not significantly interfere with the ability of the NDV LaSota strain-vectored ILTV gB and gD vaccine candidates to elicit protective immunity against infectious laryngotracheitis., (Published by Elsevier Ltd.)

Synthetic oligosaccharide-based vaccines are promising alternatives to conventional antibacterial carbohydrate vaccines prepared with isolated polysaccharides. Unlike polysaccharides, synthetic glycans are well defined, contaminant-free, and accessible even for pathogens that cannot be fermented or show limited carbohydrate biosynthesis in vitro. However, identifying synthetic glycan B cell epitopes that induce protective immunity has traditionally been a time-consuming trial-and-error process, as predicting the immunogenicity of an oligosaccharide by means of structure alone is not straightforward. We here describe how synthetic oligosaccharide epitopes for candidate vaccines can be rationally identified prior to preclinical immunogenicity studies. Epitopes are selected on the basis of their recognition by antibodies associated with protection from disease in humans or small animals. In addition, we show how murine antibody responses to a large oligosaccharide can inform the identification of a minimal B cell epitope that may help designing easy to synthesize vaccine candidates. The procedures, exemplified with a surface carbohydrate of Clostridium difficile, may serve as a guideline for selecting protective oligosaccharide epitopes for vaccines against infectious and malignant diseases., (© 2017 Elsevier Inc. All rights reserved.)

Tuberculosis continues to be a great cause of morbidity and mortality in different parts of the world. Unfortunately, the current BCG vaccine being administered is not fully protective against tuberculosis; therefore, there is a great need for alternate vaccines. With an aim to develop such vaccines, we have analyzed the utility of Bacillus subtilis spores for the expression of two major immunodominant antigens of Mycobacterium tuberculosis, Ag85B and CFP10. We created three recombinant B. subtilis strains to express a truncated fusion of Ag85B 191-325 and CFP10 1-70 antigens (T85BCFP), either on the spore coat (MTAG1 strain) or in the cytosol of B. subtilis (MTAG 2 and MTAG 3 strains). Examination of spores isolated from these strains revealed successful expression of T85BCFP antigens on the spore coat of MTAG1 as well as in the cytosol of vegetatively grown cells of MTAG2 and MTAG3, indicating that spores can indeed express M. tuberculosis antigens. In vitro antigen presentation assays with spore-infected mouse bone marrow derived macrophages (BMDM) showed that all three recombinant spores could deliver these antigens to antigen presenting cells (APCs). Mice immunized with recombinant spores displayed significantly higher levels of Ag85B specific IFN-γ producing cells in the spleen than in mice immunized with wild-type (non-recombinant) spores. In addition, these mice showed relatively higher levels of Ag85B specific IgG antibodies in the serum in comparison to mice immunized with non-recombinant spores, thus providing additional evidence that recombinant spores can deliver these antigens in vivo. These results suggest that B. subtilis spores are ideal vehicles for antigen delivery and have great potential in the development of primary and booster vaccines against tuberculosis., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The use of corn smut for the production of recombinant vaccines has been recently implemented by our group. In this study, the stability and immunogenic properties of the corn smut-based cholera vaccine, based on the cholera toxin B subunit (CTB), were determined in mouse. The immunogenic potential of distinct corn smut CTB doses ranging from 1 to 30μg were assessed, with maximum humoral responses at both the systemic (IgG) and intestinal (IgA) levels at a dose of 15μg. The humoral response last for up to 70days after the third boost. Mice were fully protected against a challenge with cholera toxin after receiving three 15μg-doses. Remarkably, the corn smut-made vaccine retained its immunogenic activity after storage at room temperature for a period of 1year and no reduction on CTB was observed following exposure at 50°C for 2h. These data support the use of the corn smut-made CTB vaccine as a highly stable and effective immunogen and justify its evaluation in target animal models, such as piglet and sheep, as well as clinical evaluations in humans., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The outbreak of the H5N1 highly pathogenic avian influenza which exhibits high variation had brought a serious threat to the safety of humanity. To overcome this high variation, hemagglutinin-based recombinant subunit vaccine with rational design has been considered as a substitute for traditional virion-based vaccine development. Here, we expressed HA1 part of the hemagglutinin protein using the Pichia pastoris expression system and attained a high yield of about 120 mg/L through the use of fed-batch scalable fermentation. HA1 protein in the culture supernatant was purified using two-step ion-exchange chromatography. The resultant HA1 protein was homogeneous in solution in a glycosylated form, as confirmed by endoglycosidase H treatment. Sedimentation velocity tests, silver staining of protein gels, and immunoblotting were used for verification. The native HA1 reacted well with conformational, cross-genotype, neutralizing monoclonal antibodies, whereas a loss of binding activity was noted with the denatured HA1 form. Moreover, the murine anti-HA1 serum exhibited a virus-capture capability in the hemagglutination inhibition assay, which suggests that HA1 harbors native-like epitopes. In conclusion, soluble HA1 was efficiently expressed and purified in this study. The functional glycosylated protein will be an alternative for the development of recombinant protein-based influenza vaccine.

Background: Chlamydia trachomatis is a human pathogen which causes a number of pathologies, including genital tract infections in women that can result in tubal infertility. Prevention of infection and disease control might be achieved through vaccination; however, a safe, efficacious and cost-effective vaccine against C. trachomatis infection remains an unmet medical need. C. trachomatis major outer membrane protein (MOMP), a β-barrel integral outer membrane protein, is the most abundant antigen in the outer membrane of the bacterium and has been evaluated as a subunit vaccine candidate. Recombinant MOMP (rMOMP) expressed in E. coli cytoplasm forms inclusion bodies and rMOMP extracted from inclusion bodies results in a reduced level of protection compared to the native MOMP in a mouse challenge model., Results: We sought to target the recombinant expression of MOMP to the E. coli outer membrane (OM). Successful surface expression was achieved with codon harmonization, utilization of low copy number vectors and promoters with moderate strength, suitable leader sequences and optimization of cell culture conditions. rMOMP was extracted from E. coli outer membrane, purified, and characterized biophysically. The OM expressed and purified rMOMP is immunogenic in mice and elicits antibodies that react to the native antigen, Chlamydia elementary body (EB)., Conclusions: C. trachomatis MOMP was functionally expressed on the surface of E. coli outer membrane. The OM expressed and purified rMOMP elicits antibodies that react to the native antigen, Chlamydia EB, in a mouse immunogenicity model. Surface expression of MOMP could provide useful reagents for vaccine research, and the methodology could serve as a platform to produce other outer membrane proteins recombinantly.

Modified Vaccinia Virus Ankara (MVA) is employed widely as an experimental vaccine vector for its lack of replication in mammalian cells and high expression level of foreign/heterologous genes. Recombinant MVAs (rMVAs) are used as platforms for protein production as well as vectors to generate vaccines against a high number of infectious diseases and other pathologies. The portrait of the virus combines desirable elements such as high-level biological safety, the ability to activate appropriate innate immune mediators upon vaccination, and the capacity to deliver substantial amounts of heterologous antigens. Recombinant MVAs encoding proteins of bluetongue virus (BTV), an Orbivirus that infects domestic and wild ruminants transmitted by biting midges of the Culicoides species, are excellent vaccine candidates against this virus. In this chapter we describe the methods for the generation of rMVAs encoding VP2, NS1, and VP7 proteins of bluetongue virus as a model example for orbiviruses. The protocols included cover the cloning of VP2, NS1, and VP7 BTV-4 genes in a transfer plasmid, the construction of recombinant MVAs, the titration of virus working stocks and the protein expression analysis by immunofluorescence and radiolabeling of rMVA infected cells as well as virus purification.

Poxvirus-vectors have been widely used in vaccine development for several important human and animal diseases; some of these vaccines have been licensed and used extensively. Swinepox virus (SPV) is well suited to develop recombinant vaccines because of its large packaging capacity for recombinant DNA, its host range specificity, and its ability to induce appropriate immune responses.

Unlabelled: Backgound and aims: Dermatophagoides peteronyssinus is one of the important house dust mites responsible for allergic asthma that can be tentatively managed by specific immunotherapy. The present study was to construct a vector encoding T-cell epitopes of major allergen group 1 of Dermatophagoides pteronyssinus as a vaccine delivered by MHC class II pathway., Methods: the nucleotide sequences of the 3 target genes were synthesized, including TAT, IhC and the recombinant fragment of Der p 1 encoding 3 T-cell epitopes. After amplification of the 3 target fragments by PCR and digestion with corresponding restriction endonucleases, the recombinant gene TAT-IhC-Der p 1-3T was ligated using T4 DNA ligase and inserted into the prokaryotic expression vector pET28a(+) to construct the recombinant plasmid pET- 28a(+)-TAT-IhC-Der p 1-3T, which was confirmed by digestion with restriction endonucleases and sequencing. The recombinant vector was transformed into E. coli strain BL21 (DE3) and induced with IPTG, and the induced protein TAT-IhC-Der p1-3T was detected by SDS-PAGE. After purification, the recombinant protein was confirmed by Western blotting and its allergenicity tested using IgE-binding assay., Results: the recombinant plasmid pET-28a-TAT-IhCDer p1-3T was successfully constructed as confirmed by restriction endonuclease digestion and sequencing, and the expression of the recombinant protein TAT-IhC-Der p1-3T was induced in E. coli. Western blotting verified successfull purification of the target protein, which showed a stronger IgE-binding ability than Der p1., Conclusion: we successfully constructed the recombinant expression vector pET-28a-TAT-IhC-Der p1-3T expressing a T-cell epitope vaccine delivered by MHC II pathway with strong IgE-binding ability, which provides a basis for further study on specific immunotherapy via MHC class II pathway., (Copyright AULA MEDICA EDICIONES 2014. Published by AULA MEDICA. All rights reserved.)

Safety of vaccines can be compromised by contamination with adventitious agents. One potential source of adventitious agents is a vaccine seed, typically derived from historic clinical isolates with poorly defined origins. Here we generated synthetic poliovirus seeds derived from chemically synthesized DNA plasmids encoding the sequence of wild-type poliovirus strains used in marketed inactivated poliovirus vaccines. The synthetic strains were phenotypically identical to wild-type polioviruses as shown by equivalent infectious titers in culture supernatant and antigenic content, even when infection cultures are scaled up to 10-25L bioreactors. Moreover, the synthetic seeds were genetically stable upon extended passaging on the PER.C6 cell culture platform. Use of synthetic seeds produced on the serum-free PER.C6 cell platform ensures a perfectly documented seed history and maximum control over starting materials. It provides an opportunity to maximize vaccine safety which increases the prospect of a vaccine end product that is free from adventitious agents., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Live attenuated strains of Salmonella typhimurium have been extensively investigated as vaccines for a number of infectious diseases. However, there is still little information available concerning aspects of their metabolism. S. typhimurium and Escherichia coli show a high degree of similarity in terms of their genome contents and metabolic networks. However, this work presents experimental evidence showing that significant differences exist in their abilities to direct carbon fluxes to biomass and energy production. It is important to study the metabolism of Salmonella to elucidate the formation of acetate and other metabolites involved in optimizing the production of biomass, essential for the development of recombinant vaccines. The metabolism of Salmonella under aerobic conditions was assessed using continuous cultures performed at dilution rates ranging from 0.1 to 0.67 h(-1), with glucose as main substrate. Acetate assimilation and glucose metabolism under anaerobic conditions were also investigated using batch cultures. Chemostat cultivations showed deviation of carbon towards acetate formation, starting at dilution rates above 0.1 h(-1). This differed from previous findings for E. coli, where acetate accumulation was only detected at dilution rates exceeding 0.4 h(-1), and was due to the lower rate of acetate assimilation by S. typhimurium under aerobic conditions. Under anaerobic conditions, both microorganisms mainly produced ethanol, acetate, and formate. A genome-scale metabolic model, reconstructed for Salmonella based on an E. coli model, provided a poor description of the mixed fermentation pattern observed during Salmonella cultures, reinforcing the different patterns of carbon utilization exhibited by these closely related bacteria., (© 2015 American Institute of Chemical Engineers.)

Recently, we developed a high yield production process for outer membrane particles from genetically modified bacteria, called Generalized Modules of Membrane Antigens (GMMA), and the corresponding simple two step filtration purification, enabling economic manufacture of these particles for use as vaccines. Using a Shigella sonnei strain that was genetically modified to produce penta-acylated lipopolysaccharide (LPS) with reduced endotoxicity and to maintain the virulence plasmid encoding for the immunodominant O antigen component of the LPS, scale up of the process to GMP pilot scale was straightforward and gave high yields of GMMA with required purity and consistent results. GMMA were formulated with Alhydrogel and were highly immunogenic in mice and rabbits. In mice, a single immunization containing 29 ng protein and 1.75 ng of O antigen elicited substantial anti-LPS antibody levels. As GMMA contain LPS and lipoproteins, assessing potential reactogenicity was a key aspect of vaccine development. In an in vitro monocyte activation test, GMMA from the production strain showed a 600-fold lower stimulatory activity than GMMA with unmodified LPS. Two in vivo tests confirmed the low potential for reactogenicity. We established a modified rabbit pyrogenicity test based on the European Pharmacopoeia pyrogens method but using intramuscular administration of the full human dose (100 μg of protein). The vaccine elicited an average temperature rise of 0.5°C within four hours after administration, which was considered acceptable and showed that the test is able to detect a pyrogenic response. Furthermore, a repeat dose toxicology study in rabbits using intramuscular (100 μg/dose), intranasal (80 μg/dose), and intradermal (10 μg/dose) administration routes showed good tolerability of the vaccine by all routes and supported its suitability for use in humans. The S. sonnei GMMA vaccine is now in Phase 1 dose-escalation clinical trials.

Background: T-cell epitopes play the important role in T-cell immune response, and they are critical components in the epitope-based vaccine design. Immunogenicity is the ability to trigger an immune response. The accurate prediction of immunogenic T-cell epitopes is significant for designing useful vaccines and understanding the immune system., Methods: In this paper, we attempt to differentiate immunogenic epitopes from non-immunogenic epitopes based on their primary structures. First of all, we explore a variety of sequence-derived features, and analyze their relationship with epitope immunogenicity. To effectively utilize various features, a genetic algorithm (GA)-based ensemble method is proposed to determine the optimal feature subset and develop the high-accuracy ensemble model. In the GA optimization, a chromosome is to represent a feature subset in the search space. For each feature subset, the selected features are utilized to construct the base predictors, and an ensemble model is developed by taking the average of outputs from base predictors. The objective of GA is to search for the optimal feature subset, which leads to the ensemble model with the best cross validation AUC (area under ROC curve) on the training set., Results: Two datasets named 'IMMA2' and 'PAAQD' are adopted as the benchmark datasets. Compared with the state-of-the-art methods POPI, POPISK, PAAQD and our previous method, the GA-based ensemble method produces much better performances, achieving the AUC score of 0.846 on IMMA2 dataset and the AUC score of 0.829 on PAAQD dataset. The statistical analysis demonstrates the performance improvements of GA-based ensemble method are statistically significant., Conclusions: The proposed method is a promising tool for predicting the immunogenic epitopes. The source codes and datasets are available in S1 File.

Ojective: To construct the engineering bacteria with recombinant plasmid expressing the multi-epitope vaccine which composed of Helicobacter pylori urea membrane channel protein (UreI), Helicobacter pylori urease B subunit (UreB) and cholera toxin B subunit (CTB), and then to study it's microbiological characteristics., Methods: The sequence contains some dominant epitopes of Helicobacter pylori UreI and UreB was designed, and ctB was added at the N-terminal, all the sequence were linked by flexible linkers. Codon optimization was done according to Escherichia coli (E. coli) BL21 (DE3) bias, the optimized sequence was designated BIB. BIB sequence was synthesized and cloned into plasmid pET28a(+). The recombinant plasmid was confirmed by restriction enzyme digestion and DNA sequencing. The recombinant protein BIB was expressed in E. coli BL21 (DE3) and analyzed by Western blot., Results: The plasmid of pET28a(+)/BIB was constructed successfully, confirmed by restriction enzyme digestion and DNA sequencing. The recombinant protein BIB with relative molecular mass about 33 x 10(3) could be produced by E. coli BL21 (DE3) and was detected by Western blot. The relative molecular mass and N-terminal amino acid sequence of BIB were 100% identity with the design., Conclution: The engineering bacteria with recombinant plasmid expressing the multi-epitope vaccine against Helicobacter pylori was constructed successfully. The recombinant protein BIB can be identified by anti-Sydney strain 1 of Helicobacter pylori (H. pylori SS1) polyclonal antibody and anti-CTB monoclonal antibody, which demonstrated that BIB has the expected antigenicity.

We demonstrated the successful optimization of a recombinant multi-subunit malaria vaccine candidate protein for production in the methylotrophic yeast Pichia pastoris by the identification and subsequent removal of two protease cleavage sites. After observing protein degradation in the culture supernatant of a fed-batch fermentation, the predominant proteolytic fragment of the secreted recombinant protein was analyzed by mass spectrometry. The MS data indicated the cleavage of an amino acid sequence matching the yeast KEX2-protease consensus motif EKRE. The cleavage in this region was completely abolished by the deletion of the EKRE motif in a modified variant. This modified variant was produced, purified, and used for immunization of rabbits, inducing high antigen specific antibody titers (2 × 10(6) ). Total IgG from rabbit immune sera recognized different stages of Plasmodium falciparum parasites in immunofluorescence assays, indicating native folding of the vaccine candidate. However, the modified variant was still degraded, albeit into different fragments. Further analysis by mass spectrometry and N-terminal sequencing revealed a second cleavage site downstream of the motif PEVK. We therefore removed a 17-amino-acid stretch including the PEVK motif, resulting in the subsequent production of the full-length recombinant vaccine candidate protein without significant degradation, with a yield of 53 mg per liter culture volume. We clearly demonstrate that the proteolytic degradation of recombinant proteins by endogenous P. pastoris proteases can be prevented by the identification and removal of such cleavage sites. This strategy is particularly relevant for the production of recombinant subunit vaccines, where product yield and stability play a more important role than for the production of a stringently-defined native sequence which is necessary for most therapeutic molecules., (© 2014 Wiley Periodicals, Inc.)

Capsular polysaccharides are the primary antigenic components involved in protective immunity against encapsulated bacterial pathogens. Although immunization of adolescents and adults with polysaccharide antigens has reduced pathogen disease burden, pure polysaccharide vaccines have proved ineffective at conferring protective immunity to infants and the elderly, age cohorts that are deficient in their adaptive immune responses to such antigens. However, T-cell-independent polysaccharide antigens can be converted into more potent immunogens by chemically coupling to a "carrier protein" antigen. Such "conjugate vaccines" efficiently induce antibody avidity maturation, isotype switching, and immunological memory in immunized neonates. These immune responses have been attributed to T-cell recognition of peptides derived from the coupled carrier protein. The covalent attachment of polysaccharide antigens to the carrier protein is thought to be imperative to the immunological properties of conjugate vaccines. Here we provide evidence that covalent attachment to carrier proteins is not required for conversion of T-independent antigens into T-dependent immunogens. Simple entrapment of polysaccharides or a d-amino acid polymer antigen in a cross-linked protein matrix was shown to be sufficient to produce potent immunogens that possess the key characteristics of conventional conjugate vaccines. The versatility and ease of manufacture of these antigen preparations, termed protein capsular matrix vaccines (PCMVs), will likely provide improvements in the manufacture of vaccines designed to protect against encapsulated microorganisms. This in turn could improve the availability of such vaccines to the developing world, which has shown only a limited capacity to afford the cost of conventional conjugate vaccines.

During the last 25 years, the technology to produce recombinant vaccines in plant cells has evolved from modest proofs of the concept to viable technologies adopted by some companies due to significant improvements in the field. Viral-based expression strategies have importantly contributed to this success owing to high yields, short production time (which is in most cases free of tissue culture steps), and the implementation of confined processes for production under GMPs. Herein the distinct expression systems based on viral elements are analyzed. This review also presents the outlook on how these technologies have been successfully applied to the development of plant-based vaccines, some of them being in advanced stages of development. Perspectives on how viral expression systems could allow for the development of innovative oral vaccines constituted by minimally-processed plant biomass are discussed.

The strain composition of influenza vaccines must be changed regularly to track influenza virus antigenic evolution. During outbreaks with pandemic potential, strain changes are urgent. The systems for accomplishing vaccine strain changes have required the shipment of viruses and other biological materials around the globe, with delays in vaccine availability, and have used legacy techniques of egg-based virus cultivation, resulting in vaccine mismatches. In collaboration with Synthetic Genomics Vaccines Inc. and the US Biomedical Advanced Research and Development Authority, Novartis has developed a synthetic approach to influenza vaccine virus generation. Synthetic influenza vaccine viruses and mammalian cell culture technology promise influenza vaccines that match circulating influenza strains more closely and are delivered in greater quantities, more rapidly than vaccines produced by conventional technologies. These new technologies could yield an improved influenza vaccine response system in which viral sequence data from many sources are posted on the Internet, are downloaded by vaccine manufacturers, and are used to rescue multiple, attenuated vaccine viruses directly on high yielding backbones. Elements of this system were deployed in the response to the 2013 H7N9 influenza outbreak in China. The result was the production, clinical testing, and stockpiling of an H7N9 vaccine before the second wave of the outbreak struck at the end of 2013. Future directions in synthetic influenza vaccine technology include the automation of influenza virus rescue from sequence data and the merger of synthetic and self-amplifying mRNA vaccine technologies. The result could be a more robust and effective influenza vaccine system.

Artificial genetic constructs that direct the synthesis of self-replicating RNA molecules are used widely to induce gene silencing, for bioproduction, and for vaccination. Interestingly, one variant of the self-replicon has not been discussed in the literature: namely, transgenic organisms that synthesise alien replicons. For example, plant cells may be easily genetically modified to produce bacteriophages or insect viruses. Alien replicon-producing organisms (ARPOs) may serve as a unique tool for biocontrol or to selectively influence the characteristics of a target organism. The ARPO approach would have to meet strict biosafety criteria, and its practical applications are problematic. However, a discussion on ARPO applicability would be valuable to outline the full set of options available in the bioengineering toolbox. In this paper, RNA replicons for bioengineering are reviewed briefly, and the ARPO approach is discussed., (© 2014 WILEY Periodicals, Inc.)

The IgE Fcε3 domain is an active immunotherapeutic target for asthma and other allergic diseases. However, previous methods for preparing IgE fusion protein vaccines are complex. Antigen 43 (Ag43) is a surface protein found in Escherichia coli that contains α and β subunits (the α subunit contains multiple T epitopes). Here we constructed a novel Ag43 surface display system (Ag43 system) to express Ag43 chimeric proteins to disrupt immune tolerance against IgE. The Ag43 system was constructed from the E. coli strain Tan109, in which the Ag43 gene was deleted and a recombinant plasmid (pETAg43) expressing a partial Ag43 gene was introduced. The Fcε3 domain of the IgE gene was then subcloned into plasmid pETAg43, resulting in a recombinant plasmid pETAg43/Fcε3, which was used to transform Tan109 for Ag43/Fcε3 surface expression. Thereafter, Ag43/Fcε3 was investigated as an asthma vaccine in a mouse model. Ag43/Fcε3 was expressed on and could be separated from the bacterial surface by heating to 60° while retaining activity. Ag43/Fcε3, as a protein vaccine, produced neutralizing autoantibodies to murine IgE, induced significant anti-asthma effects, and regulated IgE and T helper cytokines in a murine asthma model. Data show that Ag43/Fcε3 chimeric protein is a potential model vaccine for asthma treatment, and that the Ag43 system may be an effective tool for novel vaccine preparation to break immune tolerance to other self-molecules., (© 2014 John Wiley & Sons Ltd.)

The hepatitis E virus (HEV) vaccine, Hecolin(®), was licensed in China for the prevention of HEV infection and HEV-related diseases with demonstrated safety and efficacy [1,2]. The vaccine is composed of a truncated HEV capsid protein, p239, as the sole antigen encoded by open reading frame 2 and produced using Escherichia coli platform. The production of this virus-like particle (VLP) form of the antigen was successfully scaled up 50-fold from a bench scale to a manufacturing scale. Product consistency was demonstrated using a combination of biophysical, biochemical and immunochemical methods, which revealed comparable antigen characteristics among different batches. Particle size of the nanometer scale particulate antigen and presence of key epitopes on the particle surface are two prerequisites for an efficacious VLP-based vaccine. The particle size was monitored by several different methods, which showed diameters between 20 and 30nm for the p239 particles. The thermal stability and aggregation propensity of the antigen were assessed using differential scanning calorimetry and cloud point assay under heat stress conditions. Key epitopes on the particulate antigen were analyzed using a panel of murine anti-HEV monoclonal antibodies (mAbs). The immuno reactivity to the mAbs among the different antigen lots was highly consistent when analyzed quantitatively using a surface plasmon resonance technique. Using a sandwich ELISA to probe the integrity of two different epitopes in the antigen, the specific antigenicity of multiple batches was assessed to demonstrate consistency in these critical product attributes. Overall, our findings showed that the antigen production process is robust and scalable during the manufacturing of Hecolin(®)., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In order to deliver low-cost viral capsomeres from a large amount of soluble viral VP6 protein from human rotavirus, we developed and optimized a biotechnological platform in Escherichia coli. Specifically, three different expression protocols were compared, differing in their genetic constructs, ie, a simple native histidine-tagged VP6 sequence, VP6 fused to thioredoxin, and VP6 obtained with the newly described small ubiquitin-like modifier (SUMO) fusion system. Our results demonstrate that the histidine-tagged protein does not escape the accumulation in the inclusion bodies, and that SUMO is largely superior to the thioredoxin-fusion tag in enhancing the expression and solubility of VP6 protein. Moreover, the VP6 protein produced according to the SUMO fusion tag displays well-known assembly properties, as observed in both transmission electron microscopy and atomic force microscopy images, giving rise to either VP6 trimers, 60 nm spherical virus-like particles, or nanotubes a few microns long. This different quaternary organization of VP6 shows a higher level of immunogenicity for the elongated structures with respect to the spheres or the protein trimers. Therefore, the expression and purification strategy presented here - providing a large amount of the viral capsid protein in the native form with relatively simple, rapid, and economical procedures - opens a new route toward large-scale production of a more efficient antigenic compound to be used as a vaccination tool or as an adjuvant, and also represents a top-quality biomaterial to be further modified for biotechnological purposes.

Highly conserved ectodomain of influenza virus M2 protein (M2e) is an important target for the development of universal influenza vaccines. Today, the use of chemical or genetic fusion constructs have been undertaken to overcome the low immunogenicity of M2e in vaccine formulation. However, current M2e vaccines are neither orally delivered nor heat-stable. In this study, we evaluated the immune efficacy of an orally delivered recombinant M2e vaccine containing 3 molcules of M2e consensus sequence of influenza A viruses, termed RSM2e3. To accomplish this, CotB, a spore coat of Bacillus subtilis (B. subtilis), was used as a fusion partner, and heat-stable nonpathogenic B. subtilis spores were used as the carrier. Our results showed that CotB-M2e3 fusion had no effect on spore structure or function in the resultant recombinant RSM2e3 strain and that heterologous influenza virus M2e protein was successfully displayed on the surface of the recombinant RSM2e3 spore. Importantly, recombinant RSM2e3 spores elicited strong and long-term M2e-specific systemic and mucosal immune responses, completely protecting immunized mice from lethal challenge of A/PR/8/34(H1N1) influenza virus. Taken together, our study forms a solid basis for the development of a novel orally delivered and heat-stable influenza vaccine based on B. subtilis spore surface display.

Many examples of a successful application of plant-based expression systems for production of biologically active recombinant proteins exist in the literature. These systems can function as inexpensive platforms for the large scale production of recombinant pharmaceuticals or subunit vaccines. Hemagglutinin (HA) is a major surface antigen of the influenza virus, thus it is in the centre of interests of various subunit vaccine engineering programs. Large scale production of recombinant HA in traditional expression systems, such as mammalian or insect cells, besides other limitations, is expensive and time-consuming. These difficulties stimulate an ever-increasing interest in plant-based production of this recombinant protein. Over the last few years many successful cases of HA production in plants, using both transient and stable expression systems have been reported. Various forms of recombinant HA, including monomers, trimers, virus like particles (VLPs) or chimeric proteins containing its fusion with other polypeptides were obtained and shown to maintain a proper antigenicity. Immunizations of animals (mice, ferrets, rabbits or chickens) with some of these plant-derived hemagglutinin variants were performed, and their effectiveness in induction of immunological response and protection against lethal challenge with influenza virus demonstrated. Plant-produced recombinant subunit vaccines and plant-made VLPs were successfully tested in clinical trials (Phase I and II) that confirmed their tolerance and immunogenicity.

Equine influenza virus is a leading cause of respiratory disease in horses worldwide. Disease prevention is by vaccination with inactivated whole virus vaccines. Most current influenza vaccines are generated in embryonated hens' eggs. Virions are harvested from allantoic fluid and chemically inactivated. Although this system has served well over the years, the use of eggs as the substrate for vaccine production has several well-recognized disadvantages (cost, egg supply, waste disposal and yield in eggs). The aim of this study was to evaluate a baculovirus system as a potential method for producing recombinant equine influenza hemagglutinin to be used as a vaccine. The hemagglutinin ectodomain (HA1 subunit) was cloned and expressed using a baculovirus expression vector. The expression was determined by SDS-PAGE and immunoblotting. A high yield, 20μg/ml of viral protein, was obtained from recombinant baculovirus-infected cells. The immune response in BALB/c mice was examined following rHA1 inoculation. Preliminary results show that recombinant hemagglutinin expressed from baculovirus elicits a strong antibody response in mice; therefore it could be used as an antigen for subunit vaccines and diagnostic tests., (Copyright © 2013 Asociación Argentina de Microbiología. Publicado por Elsevier España. All rights reserved.)

Elicitation of broad humoral immune responses is a critical factor in the development of effective HIV vaccines. In an effort to develop low-cost candidate vaccines based on multiepitopic recombinant proteins, this study has been undertaken to assess and characterize the immunogenic properties of a lettuce-derived C4(V3)6 multiepitopic protein. This protein consists of V3 loops corresponding to five different HIV isolates, including MN, IIIB, RF, CC, and RU. In this study, both Escherichia coli and lettuce-derived C4(V3)6 have elicited local and systemic immune responses when orally administered to BALB/c mice. More importantly, lettuce-derived C4(V3)6 has shown a higher immunogenic potential than that of E. coli-derived C4(V3)6. Moreover, when reactivity of sera from mice immunized with C4(V3)6 are compared with those elicited by a chimeric protein carrying a single V3 sequence, broader responses have been observed. The lettuce-derived C4(V3)6 has elicited antibodies with positive reactivity against V3 loops from isolates MN, RF, and CC. In addition, splenocyte proliferation assays indicate that significant T-helper responses are induced by the C4(V3)6 immunogen. Taken together, these findings account for the observed elicitation of broader humoral responses by the C4(V3)6 multiepitopic protein. Moreover, they provide further validation for the production of multiepitopic vaccines in plant cells as this serves not only as a low-cost expression system, but also as an effective delivery vehicle for orally administered immunogens.

Baculovirus expression system offers the advantage of expression of several large proteins simultaneously by a single recombinant virus. To date, expression of multiple large (>100kDa) proteins has been hampered by the need to generate large constructs and repeat use of homologous sequence and promoter. The development of multi-loci baculovirus expression system overcomes these issues by enabling the recombination of large foreign sequences into different regions of the genome. In this paper, we have examined the co-expression of African horse sickness virus (AHSV) VP2 proteins from multiple serotypes in a single recombinant baculovirus. To this end, recombinant baculoviruses expressing multiple AHSV VP2 proteins were generated and it was found that up to six different AHSV serotypes (serotype 1, 3, 4, 5, 7 and 8) VP2 proteins (∼120kDa) could be expressed simultaneously from different loci of baculovirus genome. The expression of VP2 of one serotype was not significantly hindered by the presence of other serotypes, although there were slight differences in expression level between different serotypes. The expression of VP2 of further serotypes from additional loci resulted in a lesser expression level of VP2 proteins. Based on these findings, three additional recombinant baculoviruses encompassing all nine AHSV serotypes were constructed (serotypes 1, 7, 8 or serotypes 2, 4, 5 or serotypes 3, 6, 9) and each of the triple recombinant viruses exhibited similar expression level of each VP2. This system allows for the expression of a number of large proteins that has the potential to be exploited for multivalent vaccines production., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Taenia solium is a zoonotic parasite that causes cysticercosis. The parasite is a major cause of human disease in impoverished communities where it is transmitted to humans from pigs which act as intermediate hosts. Vaccination of pigs to prevent transmission of T. solium to humans is an approach that has been investigated to control the disease. A recombinant vaccine antigen, TSOL18, has been remarkably successful at reducing infection of pigs with T. solium in several experimental challenge trials. The vaccine has been shown to eliminate transmission of naturally acquired T. solium in a field trial conducted in Africa. We recently reported that the vaccine was also effective in a field trial conducted in Peru. The TSOL18 recombinant antigen for each of these trials has been produced by expression in Escherichia coli. Here we discuss research that has been undertaken on the TSOL18 antigen and related antigens with a focus on improved methods of preparation of recombinant TSOL18 and optimized expression in Escherichia coli.

Because of the importance of carbohydrate-protein interactions in biological processes, the development of glycoclusters and glycodendrimers capable of mimicking the multivalent display of carbohydrates at the cell surface has become a major field of research over the last decade. Among the large variety of scaffolds that are now available, peptides and cyclopeptides are widely used for the multivalent presentation of glycans. This review will provide an overview of the most recent advances in the preparation and utilization of linear glycopeptides and glycocyclopeptides in glycobiology.

Are there general rules to obtain efficient immunization against carbohydrate antigens? Thanks to technological advances in glycobiology and glycochemistry we entered a new era in which the rational design of carbohydrate vaccines has become an achievable goal. The aim of this Tutorial Review is to present the most recent accomplishments in the field of semi and fully synthetic carbohydrate vaccines against viruses, bacteria and cancer. It is also pointed out that the understanding of the chemical and biochemical processes related to immunization allows the modern chemist to rationally design carbohydrate vaccines with improved efficiency.

Infectious Bursal Disease Virus (IBDV) is the etiological agent of an immunosuppressive and highly contagious disease that affects young birds. This disease causes important economic losses in the poultry industry worldwide. The VP2 protein has been used for the development of subunit vaccines in a variety of heterologous platforms. In this context, the aim of this study was to investigate VP2 expression and immunogenicity using an experimental plant-based vaccine against IBDV. We determined that the agroinfiltration of N. benthamiana leaves allowed the production of VP2 with no apparent change on its conformational epitopes. Chickens intramuscularly immunized in a dose/boost scheme with crude concentrated extracts developed a specific humoral response with viral neutralizing ability. Given these results, it seems plausible for a plant-based vaccine to have a niche in the veterinary field. Thus, plants can be an adequate system of choice to produce immunogens against IBDV., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Malaria is a widespread and infectious disease that is a leading cause of death in many parts of the world. Eradication of malaria has been a major world health goal for decades, but one that still remains elusive. Other diseases have been eradicated using vaccination, but traditional vaccination methods have thus far been unsuccessful for malaria. Infection by Plasmodium species, the causative agent of malaria, is currently treated with drug-based therapies, but an increase in drug resistance has led to the need for new methods of treatment. A promising strategy for malaria treatment is to combine transmission blocking vaccines (TBVs) that prevent spread of disease with drug-based therapies to treat infected individuals. TBVs can be developed against surface protein antigens that are expressed during parasite reproduction in the mosquito. When the mosquito ingests blood from a vaccinated individual harboring the Plasmodium parasite, the antibodies generated by vaccination prevent completion of the parasites life-cycle. Animal studies have shown that immunization with Pfs48/45 results in the production of malaria transmission blocking antibodies; however, the development of this vaccine candidate has been hindered by poor expression in both prokaryotic and eukaryotic hosts. Recently, the chloroplast of Chlamydomonas reinhardtii has been used to express complex recombinant proteins. In this study, we show that the C-terminal antigenic region of the Pfs48/45 antigen can be expressed in the chloroplast of the green algae C. reinhardtii and that this recombinant protein has a conformation recognized by known transmission blocking antibodies. Production of this protein in algae has the potential to scale to the very large volumes required to meet the needs of millions at risk for contracting malaria.

Monoclonal antibodies, modern vaccines and gene therapy have become a major field in modern biotechnology, especially in the area of human health and fascinating developments achieved in the past decades are impressive examples of an interdisciplinary interplay between medicine, biology and engineering. Among the classical products from cells one can find viral vaccines, monoclonal antibodies, and interferons, as well as recombinant therapeutic proteins. Gene therapy opens up challenging new areas. In this review, a definitions of these processes are given and fields of application and products, as well as the future prospects, are discussed.

Introduction: Cholera toxin B subunit (CTB) is a component of an internationally licensed oral cholera vaccine. The protein induces neutralizing antibodies against the holotoxin, the virulence factor responsible for severe diarrhea. A field clinical trial has suggested that the addition of CTB to killed whole-cell bacteria provides superior short-term protection to whole-cell-only vaccines; however, challenges in CTB biomanufacturing (i.e., cost and scale) hamper its implementation to mass vaccination in developing countries. To provide a potential solution to this issue, we developed a rapid, robust, and scalable CTB production system in plants., Methodology/principal Findings: In a preliminary study of expressing original CTB in transgenic Nicotiana benthamiana, the protein was N-glycosylated with plant-specific glycans. Thus, an aglycosylated CTB variant (pCTB) was created and overexpressed via a plant virus vector. Upon additional transgene engineering for retention in the endoplasmic reticulum and optimization of a secretory signal, the yield of pCTB was dramatically improved, reaching >1 g per kg of fresh leaf material. The protein was efficiently purified by simple two-step chromatography. The GM1-ganglioside binding capacity and conformational stability of pCTB were virtually identical to the bacteria-derived original B subunit, as demonstrated in competitive enzyme-linked immunosorbent assay, surface plasmon resonance, and fluorescence-based thermal shift assay. Mammalian cell surface-binding was corroborated by immunofluorescence and flow cytometry. pCTB exhibited strong oral immunogenicity in mice, inducing significant levels of CTB-specific intestinal antibodies that persisted over 6 months. Moreover, these antibodies effectively neutralized the cholera holotoxin in vitro., Conclusions/significance: Taken together, these results demonstrated that pCTB has robust producibility in Nicotiana plants and retains most, if not all, of major biological activities of the original protein. This rapid and easily scalable system may enable the implementation of pCTB to mass vaccination against outbreaks, thereby providing better protection of high-risk populations in developing countries.

Plants have a demonstrated potential for large-scale, rapid production of recombinant proteins for diverse product applications, including subunit vaccines and monoclonal antibodies. In this field, the accent has recently shifted from the engineering of "edible" vaccines based on stable expression of target protein in transgenic or transplastomic plants to the development of purified formulated vaccines that are delivered via injection. The injectable vaccines are commonly produced using transient expression of target gene delivered into genetically unmodified plant host via viral or bacterial vectors. Most viral vectors are based on plant RNA viruses, where nonessential sequences are replaced with the gene of interest. Utilization of viral hybrids that consist of genes and regulatory elements of different virus species, or transcomplementation systems (vector/transgene) had a substantial impact on the level of target protein expression. Development and introduction of agroviral hybrid vectors that combine genetic elements of bacterial binary plasmids and plant viral vectors, and agroinfiltration as a tool of the vector delivery have resulted in significant progress in large-scale production of recombinant vaccines and monoclonal antibodies in plants. This article presents an overview of plant hybrid viral vector expression systems developed so far.

Vaccines are the most efficient and cost-effective means of preventing infectious disease. However, traditional vaccine approaches have thus far failed to provide protection against human immunodeficiency virus (HIV), tuberculosis, malaria, and many other diseases. New approaches to vaccine development are needed to address some of these intractable problems. In this report, we review the literature identifying stimulatory effects of mesenchymal stem cells (MSC) on immune responses and explore the potential for MSC as a novel, universal vaccination platform. MSC are unique bone marrow-derived multipotent progenitor cells that are presently being exploited as gene therapy vectors for a variety of conditions, including cancer and autoimmune diseases. Although MSC are predominantly known for anti-inflammatory properties during allogeneic MSC transplant, there is evidence that MSC can actually promote adaptive immunity under certain settings. MSC have also demonstrated some success in anti-cancer therapeutic vaccines and anti-microbial prophylactic vaccines, as we report, for the first time, the ability of modified MSC to express and secrete a viral antigen that stimulates antigen-specific antibody production in vivo. We hypothesize that the unique properties of modified MSC may enable MSC to serve as an unconventional but innovative, vaccine platform. Such a platform would be capable of expressing hundreds of proteins, thereby generating a broad array of epitopes with correct post-translational processing, mimicking natural infection. By stimulating immunity to a combination of epitopes, it may be possible to develop prophylactic and even therapeutic vaccines to tackle major health problems including those of non-microbial and microbial origin, including cancer, or an infectious disease like HIV, where traditional vaccination approaches have failed.