David E. Clements

Development of a recombinant tetravalent dengue virus vaccine: immunogenicity and efficacy studies in mice and monkeys.

Truncated recombinant dengue virus envelope protein subunits (80E) are efficiently expressed using the Drosophila Schneider-2 (S2) cell expression system. Binding of conformationally sensitive antibodies as well as X-ray crystal structural studies indicate that the recombinant 80E subunits are properly folded native-like proteins. Combining the 80E subunits from each of the four dengue serotypes with ISCOMATRIX adjuvant, an adjuvant selected from a set of adjuvants tested for maximal and long lasting immune responses, results in high titer virus neutralizing antibody responses. Immunization of mice with a mixture of all four 80E subunits and ISCOMATRIX adjuvant resulted in potent virus neutralizing antibody responses to each of the four serotypes. The responses to the components of the tetravalent mixture were equivalent to the responses to each of the subunits administered individually. In an effort to evaluate the potential protective efficacy of the Drosophila expressed 80E, the dengue serotype 2 (DEN2-80E) subunit was tested in both the mouse and monkey challenge models. In both models protection against viral challenge was achieved with low doses of antigen in the vaccine formulation. In non-human primates, low doses of the tetravalent formulation induced good virus neutralizing antibody titers to all four serotypes and protection against challenge with the two dengue virus serotypes tested. In contrast to previous reports, where subunit vaccine candidates have generally failed to induce potent, protective responses, native-like soluble 80E proteins expressed in the Drosophila S2 cells and administered with appropriate adjuvants are highly immunogenic and capable of eliciting protective responses in both mice and monkeys. These results support the development of a dengue virus tetravalent vaccine based on the four 80E subunits produced in the Drosophila S2 cell expression system.

The Development of Recombinant Subunit Envelope-Based Vaccines to Protect Against Dengue Virus Induced Disease

Challenges associated with the interference observed between the dengue virus components within early tetravalent live-attenuated vaccines led many groups to explore the development of recombinant subunit based vaccines. Initial efforts in the field were hampered by low yields and/or improper folding, but the use of the Drosophila S2 cell expression system provided a mechanism to overcome these limitations. The truncated dengue envelope proteins (DEN-80E) for all four dengue virus types are expressed in the S2 system at high levels and have been shown to maintain native-like conformation. The DEN-80E proteins are potent immunogens when formulated with a variety of adjuvants, inducing high titer virus neutralizing antibody responses and demonstrating protection in both mouse and non-human primate models. Tetravalent vaccine formulations have shown no evidence of immune interference between the four DEN-80E antigens in preclinical models. Based on the promising preclinical data, the recombinant DEN-80E proteins have now advanced into clinical studies. An overview of the relevant preclinical data for these recombinant proteins is presented in this review.

Dengue vaccines: progress and challenges

With several dengue vaccine candidates progressing through clinical trials, several options for controlling this disease appear feasible. This would represent a major achievement and reflect decades of research and development activities. The challenges associated with the limited understanding of protective responses and those factors which determine disease severity remain, but with prospective studies ongoing in various dengue endemic areas and the initiation of dengue vaccine efficacy trials, immune responses are being evaluated in the context of protection and severe disease and these studies are highly likely to provide additional insights.

Use of Recombinant Envelope Proteins for Serological Diagnosis of Dengue Virus Infection in an Immunochromatographic Assay

ABSTRACT An immunochromatographic test that incorporates recombinant antigens (Dengue Duo Rapid Strip Test; PanBio, Brisbane, Australia) has recently become commercially available. This assay is performed in 15 min and detects both immunoglobulin M (IgM) and IgG in a capture format. The four recombinant proteins used represent the N-terminal 80% of the viral envelope glycoproteins of dengue viruses 1, 2, 3, and 4, respectively. The sensitivity and specificity of the recombinant-antigen-based assay were 90 and 86%, respectively. The similar diagnostic performance of these antigens to that of enzyme-linked immunosorbent assays using whole dengue virus suggests that they mimic whole dengue viruses in primary structure and epitope conformation. These results suggest that recombinant proteins can be used in diagnostic assays for dengue to overcome safety issues associated with the use of whole virus.

Multiple copies of a DNA sequence from Pseudomonas syringae pathovar phaseolicola abolish thermoregulation of phaseolotoxin production

Phaseolotoxin, a phytotoxin of Pseudomonas syringae pv. phaseolicola, is produced at 18°C but not at 28°C. Here we report that a fragment (24.4 kb) cloned from the wild‐type strain, which does not harbour a gene(s) involved in phaseolotoxin biosynthesis, abolishes this thermoregulation in the wild type and suppresses a Tox− mutant at both temperatures. A subclone harbouring a 465bp fragment contains motifs that are characteristic of DNA‐binding sites. In mobility shift assays we have detected a protein(s) from the wild‐type and the mutant strains, grown at appropriate temperatures, that specifically binds to the fragment containing the DNA‐binding motifs. We propose that the binding protein is a repressor which is ‘titrated’ by this fragment when it is present in the cell on a multiple copy plasmid, thus allowing expression of phaseolotoxin genes.

Preclinical development of a dengue tetravalent recombinant subunit vaccine: Immunogenicity and protective efficacy in nonhuman primates

We describe here the preclinical development of a dengue vaccine composed of recombinant subunit carboxy-truncated envelope (E) proteins (DEN-80E) for each of the four dengue serotypes. Immunogenicity and protective efficacy studies in Rhesus monkeys were conducted to evaluate monovalent and tetravalent DEN-80E vaccines formulated with ISCOMATRIX™ adjuvant. Three different doses and two dosing regimens (0, 1, 2 months and 0, 1, 2, and 6 months) were evaluated in these studies. We first evaluated monomeric (DEN4-80E) and dimeric (DEN4-80EZip) versions of DEN4-80E, the latter generated in an attempt to improve immunogenicity. The two antigens, evaluated at 6, 20 and 100 μg/dose formulated with ISCOMATRIX™ adjuvant, were equally immunogenic. A group immunized with 20 μg DEN4-80E and Alhydrogel™ induced much weaker responses. When challenged with wild-type dengue type 4 virus, all animals in the 6 and 20 μg groups and all but one in the DEN4-80EZip 100 μg group were protected from viremia. Two out of three monkeys in the Alhydrogel™ group had breakthrough viremia. A similar study was conducted to evaluate tetravalent formulations at low (3, 3, 3, 6 μg of DEN1-80E, DEN2-80E, DEN3-80E and DEN4-80E respectively), medium (10, 10, 10, 20 μg) and high (50, 50, 50, 100 μg) doses. All doses were comparably immunogenic and induced high titer, balanced neutralizing antibodies against all four DENV. Upon challenge with the four wild-type DENV, all animals in the low and medium dose groups were protected against viremia while two animals in the high-dose group exhibited breakthrough viremia. Our studies also indicated that a 0, 1, 2 and 6 month vaccination schedule is superior to the 0, 1, and 2 month schedule in terms of durability. Overall, the subunit vaccine was demonstrated to induce strong neutralization titers resulting in protection against viremia following challenge even 8-12 months after the last vaccine dose.

Preclinical and clinical development of a dengue recombinant subunit vaccine

This review focuses on a dengue virus (DENV) vaccine candidate based on a recombinant subunit approach which targets the DENV envelope glycoprotein (E). Truncated versions of E consisting of the N-terminal portion of E (DEN-80E) have been expressed recombinantly in the Drosophila S2 expression system and shown to have native-like conformation. Preclinical studies demonstrate that formulations containing tetravalent DEN-80E adjuvanted with ISCOMATRIX™ adjuvant induce high titer virus neutralizing antibodies and IFN-γ producing T cells in flavivirus-naïve non-human primates. The preclinical data further suggest that administration of such formulations on a 0, 1, 6 month schedule may result in higher maximum virus neutralizing antibody titers and better durability of those titers compared to administration on a 0, 1, 2 month schedule. In addition, the virus neutralizing antibody titers induced by adjuvanted tetravalent DEN-80E compare favorably to the titers induced by a tetravalent live virus comparator. Furthermore, DEN-80E was demonstrated to be able to boost virus neutralizing antibody titers in macaques that have had a prior DENV exposure. A monovalent version of the vaccine candidate, DEN1-80E, was formulated with Alhydrogel™ and studied in a proof-of-principle Phase I clinical trial by Hawaii Biotech, Inc. (NCT00936429). The clinical trial results demonstrate that both the 10 μg and 50 μg formulations of DEN1-80E with 1.25 mg of elemental aluminum were immunogenic when administered in a 3-injection series (0, 1, 2 months) to healthy, flavivirus-naïve adults. The vaccine formulations induced DENV-1 neutralizing antibodies in the majority of subjects, although the titers in most subjects were modest and waned over time. Both the 10 μg DEN1-80E and the 50 μg DEN1-80E formulations with Alhydrogel™ were generally well tolerated.

Expression vectors for Neurospora crassa and expression of a bovine preprochymosin cDNA

The filamentous fungi, owing to their ability to secrete high levels of proteins, are attractive organisms for the expression and secretion of heterologous proteins of commercial and medical value. We report the construction of three expression vectors for the production of heterologous proteins in Neurospora crassa and demonstrate their utility by expression of a bovine preprochymosin cDNA and secretion of processed, enzymatically active bovine chymosin. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. Authors Eileen T. Nakano, Raymond D. Fox, David E. Clements, Ken Koo, W. Dorsey Stuart, and John M. Ivy This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol40/iss1/19 Expression vectors for Neurospora crassa and expression of a bovine preprochymosin cDNA Eileen T. Nakano, Raymond D. Fox, David E. Clements, Ken Koo, W. Dorsey Stuart*, and John M. Ivy Hawaii Biotechnology Group, Inc., Aiea, HI 96701 and *Department of Genetics, University of Hawaii (present addresses: R.D.F. Icos, Bothel WA, 98201; K.K. Hitachi Chemical Research Center, Irvine, CA 92715) The filamentous fungi, owing to their ability to secrete high levels of proteins, are attractive organisms for the expression and secretion of heterologous proteins of commercial and medical value. We report the construction of three expression vectors for the production of heterologous proteins in Neurospora crassa and demonstrate their utility by expression of a bovine preprochymosin cDNA and secretion of processed, enzymatically active bovine chymosin. One of the three vectors is based on the constitutive transcriptional promoter and terminator of the beta-tubulin gene (Orbach et al. 1988. Mol. Cell. Biol. 8:2111-2118), and the other two incorporate the glucose repressible promoter and the terminator of grg-1 (McNally and Free 1989. Curr. Genet. 14:545-551). Results on the different levels of chymosin expression are presented. The beta-tubulin promoter vector, pTPT1, was constructed in pTZ18R (Pharmacia), in which several of the multiple cloning sites were deleted. The beta-tubulin promoter and terminator fragments were subcloned from pSV50 (Vollmer and Yanofsky 1986. Proc. Natl. Acad. Sci. USA 83:4869-4873), which expresses a mutant, benomyl resistant beta-tubulin allele. A 350 base pair SalI-SfaNI promoter fragment, ending five nucleotides upstream of the translation initiating ATG, and a 380 bp beta-tubulin terminator fragment, from an ExoIII-generated end 73 nucleotides upstream of the beta-tubulin stop codon to the downstream genomic HindIII site, were combined with KpnI, SmaI and BamHI sites between them. The two grg-1 promoter vectors are based on a genomic clone of grg-1 into which an XhoI linker had been inserted (pMTF52, gift of S. Free, State University of New York at Buffalo) 67 nucleotides downstream of the primary site of transcriptional initiation (22 nucleotides upstream of the first ATG codon). A second XhoI linker was inserted into an NaeI site downstream of the last grg-1 exon and upstream of the polyadenylation signal, and all sequences between the two XhoI sites were deleted. One version of the grg-1 promoter expression vector, pGRGS, has an 837 bp promoter fragment measured from the first site of transcription initiation, and the other, pGRGL, has a 1563 bp promoter fragment. A bovine preprochymosin cDNA (pBC8, gift of M. McCaman, Berlex) was subcloned into the three expression vectors and pMTF52. After initially subcloning the cDNA into pTZ18R, the cDNA was inserted into pTPT1 between the KpnI and BamHI sites and into pGRGS, pGRGL, and pMTF52 at the unique XhoI site generating pTCT1, pGRGSC, pGRGLC and pGRC52 (Figure 1), respectively. Chymosin, an aspartyl protease, is secreted as a zymogen, prochymosin, which is autocatalytically activated to chymosin at low pH. The enzyme is naturally found in the Published by New Prairie Press, 2017 fourth stomach of the calf and cleaves kappa-casein. We took advantage of this milk clotting activity to rapidly screen transformants for expression and secretion of chymosin. Figure 1. Neurospora crassa expression vectors TCT1, pGRGSC, and pGRGLC. Steps in the construction of the chymosin expression plasmids are described in the text. Presented are the expression cassettes for each plasmid. Stippled boxes represent the respective promoter and terminator sequences (beta-tubulin or grg-1). Open arrow represents preprochymosin sequences. We co-transformed the his-2 mtr strain of N. crassa (Stuart and Koo 1988. Genome 30:198-203) with either pTCT1, pGRGSC, pGRGLC, or pGRC52 and pSV50cosmid 6:11E, bearing his-2+, from the ordered cosmid library of Vollmer and Yanofsky (ibid). Histidine prototrophs were selected on minimal medium, and transformants were transferred to agar slants. Conidia from isolated transformants were inoculated into 5 ml of Vogels + 2% sucrose liquid medium. Following three to four days incubation, the medium was screened for presence of milk clotting activity (Ward et al. 1990. Bio/Techniques 8:436-440). Quantitation of chymosin levels from selected transformants was determined by comparison of bovine chymosin (Sigma) and recombinant chymosin on Western transfers (Figure 2). Secreted proteins from four-to-five-day-old cultures were concentrated by ultrafiltration (Centricon 30, Amicon), and Western transfers were probed with a rabbit anti-prochymosin antiserum (gift of M. McCaman, Berlex). An immunoreactive protein comigrating with bovine chymosin was observed in the culture medium of most milkclotting transformants (Fig. 2). In some instances, a higher molecular weight, immunoreactive protein was observed (e.g. see Fig. 2A, lanes 3 and 4) that might represent prochymosin or pseudochymosin. http://newprairiepress.org/fgr/vol40/iss1/19 DOI: 10.4148/1941-4765.1410 Figure 2. Western transfer of medium from various N. crassa transformants. A. Lane 1. Molecular weight markers. Phosphorylase B, 106 kD; Bovine serum albumin 80 kD; Ovalbumin 49.5 kD; Carbonic anhydrase 32.5 kD; Soybean trypsin inhibitor 27.5 kD; Lysozyme 18.5 kD. Lane 2. 100 ng authentic bovine chymosin standard. Lane 3. Concentrated medium (equivalent to 210 l of unconcentrated medium) from a pTCT1 transformant. B. Lanes 1-5: authentic bovine chymosin standards, 100 ng, 33.3 ng, 11.1 ng, 3.7 ng, 1.2 ng, respectively. Lanes 6-13: 12 l medium from individual transformants. Lanes 6-10: pGRGSC transformants. Lanes 11-13: pGRGLC transformants. (arrow) mature chymosin A range of secreted chymosin levels as assessed by milk clotting activity was observed among the various sets of transformants. As determined by Western transfer analysis for the highest expressors from each set, the non-regulated beta-tubulin promoter vector and pGRGS expressed approximately the same level of chymosin, while the longer grg-1 promoter vector, pGRGL, produced more. We estimated that the highest expressing pTCT1 transformant expresses between 0.3-0.5 ug/ml of enzymatically active chymosin. The highest expressing pGRGLC transformant secretes between 0.9-1.2 ug/ml enzymatically active recombinant bovine chymosin. The authenticity of the N. crassa-expressed chymosin was evaluated by immunoprecipitation. The anti-prochymosin serum was added to 1 ml of culture medium from a five day culture of a chymosin transformant, the mixture was incubated overnight at 4 C, and fixed Staphylococcus aureus cells were added to remove the antibody-antigen complexes. This treatment removed essentially all milk clotting activity. In addition, milk clotting was not inhibited by the serine protease inhibitor phenylmethysulfonyl fluoride. Summary: We have constructed three expression vectors based on the constitutive beta-tubulin promoter and the regulatable grg-1 promoter. We have confirmed the effectiveness of these vectors by the expression and secretion of the enzymatically active mammalian protein bovine chymosin. The chymosin was expressed from a bovine preprochymosin cDNA, and chymosin was secreted under the direction of its own secretion signal. Very little chymosin was detected in cellular protein extracts, indicating the efficiency of this heterologous secretion signal peptide in Neurospora. This is in contrast to its poor effectiveness in directing secretion of chymosin from Saccharomyces cerevisiae (Smith et al. 1985. Science 229:1219-1224) and Aspergillus (Ward et al. 1990. Bio/Techniques 8:436-440). However, secretion signal efficiency is highly variable and often appears to be protein specific. We might therefore expect a homologous fungal secretion Published by New Prairie Press, 2017 signal sequence to direct secretion of some proteins better than a heterologous secretion signal sequence. To that end, we are cloning cDNAs of selected Neurospora proteins. These expression vectors may be useful for the overexpression and study of homologous proteins and for the expression of heterologous proteins in N. crassa. Neurospora crassa naturally secretes few proteins, which may simplify purification of heterologous proteins targeted for secretion and engineered for overexpression. Neurospora crassa, therefore, has potential for development as a safe and well understood production organism. Acknowledgements: We thank W. McCaman of Berlex for preprochymosin cDNAs and antiprochymosin antiserum, S. Free for plasmid pMTF52, and Fugen Tülgar for technical assistance. This research was supported in part by a National Science Foundation Small Business Innovation Research Grant ISI-8860389 and by funds from Miki & Co., Ltd. and the Nippon Synthetic Chemical Industry Co., Ltd. http://newprairiepress.org/fgr/vol40/iss1/19 DOI: 10.4148/1941-4765.1410

Recombinant proteins of Zaire ebolavirus induce potent humoral and cellular immune responses and protect against live virus infection in mice

Infections with filoviruses in humans are highly virulent, causing hemorrhagic fevers which result in up to 90% mortality. In addition to natural infections, the ability to use these viruses as bioterrorist weapons is of significant concern. Currently, there are no licensed vaccines or therapeutics available to combat these infections. The pathogenesis of disease involves the dysregulation of the hosts immune system, which results in impairment of the innate and adaptive immune responses, with subsequent development of lymphopenia, thrombocytopenia, hemorrhage, and death. Questions remain with regard to the few survivors of infection, who manage to mount an effective adaptive immune response. These questions concern the humoral and cellular components of this response, and whether such a response can be elicited by an appropriate prophylactic vaccine. The data reported herein describe the production and evaluation of a recombinant subunit Ebola virus vaccine candidate consisting of insect cell expressed Zaire ebolavirus (EBOV) surface glycoprotein (GP) and the matrix proteins VP24 and VP40. The recombinant subunit proteins are shown to be highly immunogenic in mice, yielding both humoral and cellular responses, as well as highly efficacious, providing up to 100% protection against a lethal challenge with live virus. These results demonstrate proof of concept for such a recombinant non-replicating vaccine candidate in the mouse model of EBOV which helps to elucidate immune correlates of protection and warrants further development.

A combination of TLR-4 agonist and saponin adjuvants increases antibody diversity and protective efficacy of a recombinant West Nile Virus antigen

Members of the Flaviviridae family are the leading causes of mosquito-borne viral disease worldwide. While dengue virus is the most prevalent, the recent Zika virus outbreak in the Americas triggered a WHO public health emergency, and yellow fever and West Nile viruses (WNV) continue to cause regional epidemics. Given the sporadic nature of flaviviral epidemics both temporally and geographically, there is an urgent need for vaccines that can rapidly provide effective immunity. Protection from flaviviral infection is correlated with antibodies to the viral envelope (E) protein, which encodes receptor binding and fusion functions. TLR agonist adjuvants represent a promising tool to enhance the protective capacity of flavivirus vaccines through dose and dosage reduction and broadening of antiviral antibody responses. This study investigates the ability to improve the immunogenicity and protective capacity of a promising clinical-stage WNV recombinant E-protein vaccine (WN-80E) using a novel combination adjuvant, which contains a potent TLR-4 agonist and the saponin QS21 in a liposomal formulation (SLA-LSQ). Here, we show that, in combination with WN-80E, optimized SLA-LSQ is capable of inducing long-lasting immune responses in preclinical models that provide sterilizing protection from WNV challenge, reducing viral titers following WNV challenge to undetectable levels in Syrian hamsters. We have investigated potential mechanisms of action by examining the antibody repertoire generated post-immunization. SLA-LSQ induced a more diverse antibody response to WNV recombinant E-protein antigen than less protective adjuvants. Collectively, these studies identify an adjuvant formulation that enhances the protective capacity of recombinant flavivirus vaccines.West Nile Virus: adjuvant combinations boost vaccine efficacyThere is currently no approved human vaccine for West Nile Virus (WNV); however, it is known that protective immune responses are generally directed to the viral E protein. Neal Van Hoeven and colleagues at the Infectious Disease Research Institute in Seattle use recombinant WNV E-protein antigen adjuvanted with different combinations of a synthetic Toll-like receptor 4 agonist (SLA) and the saponin QS21 to determine optimal vaccination strategies in preclinical mouse and hamster models. SLA plus QS21 synergize in the production of neutralizing antibodies and when used together generate higher antibody diversity—a property that directly correlates with their protective capacity in vivo. Uniquely, the combination of QS21 plus SLA was also able to elicit robust T helper 1 responses. These findings highlight a promising adjuvant combination that might form the basis of an effective human WNV vaccine.