A Novel Synthetic TLR-4 Agonist Adjuvant Increases the Protective Response to a Clinical-Stage West Nile Virus Vaccine Antigen in Multiple Formulations

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Abstract

West Nile virus (WNV) is a mosquito-transmitted member of the Flaviviridae family that has emerged in recent years to become a serious public health threat. Given the sporadic nature of WNV epidemics both temporally and geographically, there is an urgent need for a vaccine that can rapidly provide effective immunity. Protection from WNV infection is correlated with antibodies to the viral envelope (E) protein, which encodes receptor binding and fusion functions. Despite many promising E-protein vaccine candidates, there are currently none licensed for use in humans. This study investigates the ability to improve the immunogenicity and protective capacity of a promising clinical-stage WNV recombinant E-protein vaccine (WN-80E) by combining it with a novel synthetic TLR-4 agonist adjuvant. Using the murine model of WNV disease, we find that inclusion of a TLR-4 agonist in either a stable oil-in-water emulsion (SE) or aluminum hydroxide (Alum) formulation provides both dose and dosage sparing functions, whereby protection can be induced after a single immunization containing only 100 ng of WN-80E. Additionally, we find that inclusion of adjuvant with a single immunization reduced viral titers in sera to levels undetectable by viral plaque assay. The enhanced protection provided by adjuvanted immunization correlated with induction of a Th1 T-cell response and the resultant shaping of the IgG response. These findings suggest that inclusion of a next generation adjuvant may greatly enhance the protective capacity of WNV recombinant subunit vaccines, and establish a baseline for future development.

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Lineage 1 and 2 Strains of Encephalitic West Nile Virus, Central Europe

Tamás Bakonyi, Éva Ivanics, Károly Erdélyi … (2006)

Geographically, West Nile virus (WNV) is the most widespread member of the Japanese encephalitis virus (JEV) complex within the genus Flavivirus and the family Flaviviridae. The first strain (B 956) was isolated from a human patient in the West Nile district of Uganda in 1937; later the virus was also detected in several mosquito species, horses, humans, and other hosts in Africa, Europe, Asia, and Australia (where it has been named Kunjin virus) ( 1 – 3 ). WNV was introduced into the United States in 1999, and it spread quickly over large parts of North America and reached Mexico ( 4 – 7 ). The clinical impact of WNV varies in different regions. In the Old World, WNV causes relatively mild infections with influenzalike symptoms or no apparent disease ( 2 ); encephalitis and fatalities in the human population, horses, or poultry are spasmodic ( 3 , 8 , 9 ). In the New World, WNV exhibits increased virulence among the local wild bird populations and causes more frequent severe central nervous system symptoms and deaths in humans and horses ( 6 , 10 ). Although exactly how WNV was introduced into New York is unclear, phylogenetic comparison of the viral nucleic acid sequences has shown a close relationship between the American WNV isolates and strains isolated from encephalitic geese and storks in Israel in 1998 ( 11 – 13 ). Experimental infections of rodents indicated that the neurovirulence of WNV correlates with its genotype, and the North American strains are highly neurovirulent for mice ( 14 ). WNV shows relatively high levels of sequence diversity. Comprehensive studies on the phylogenetic relatedness of WNV strains show that they form at least 2 main lineages ( 15 – 17 ). Lineage 1 is composed of WNV strains from different geographic regions, and it is subdivided into at least 3 clades. Clade A contains strains from Europe, Africa, the Middle East, and America; clade B represents the Australian (Kunjin) strains; and clade C contains Indian WNV isolates. Lineage 2 contains the B 956 prototype strain and other strains isolated so far exclusively in sub-Saharan Africa and Madagascar. In addition to the 2 major WNV lineages, we recently proposed 2 lineages for viruses that exhibited considerable genetic differences to the known WNV lineages: lineage 3 consists of a virus strain isolated from Culex pipiens mosquitoes at the Czech Republic/Austria border (named Rabensburg virus), and lineage 4 consists of a unique virus isolated in the Caucasus. These 2 viruses, however, may also be considered independent flaviviruses within the JEV complex ( 18 ). WNV has been known to be present in central Europe for a long time. Seroprevalence in humans was reported in several countries, including Hungary, and WNV strains were isolated from mosquitoes, humans, migrating birds, and rodents during the last 30 years ( 3 ). Until 2003, however, WNV infections in Hungary have never been associated with clinical symptoms, although a severe outbreak of West Nile encephalitis in humans was reported in 1996 and 1997 in neighboring Romania. In late summer 2003, an outbreak of encephalitis emerged in a Hungarian goose flock, resulting in a 14% death rate among 6-week-old geese (Anser anser domesticus). Based on histopathologic alterations, serologic investigations, and nucleic acid detection by reverse transcription–polymerase chain reaction (RT-PCR), WNV was diagnosed as the cause of the disease ( 19 ). Chronologically and geographically related to the outbreak in geese, a serologically confirmed WNV outbreak was also observed in humans, which involved 14 cases of mild encephalitis and meningitis ( 20 ). One year later, in August 2004, a goshawk (Accipiter gentilis) fledgling showed central nervous system symptoms and died in a national park in southeastern Hungary. When histopathologic methods and RT-PCR were used, WNV antigen and nucleic acid were detected in the organs of the bird. Furthermore, the virus was isolated after injection of suckling mice. Here we report the sequencing and phylogenetic results of these 2 encephalitic WNV strains that emerged recently in central Europe. Materials and Methods Brain specimens from one 6-week-old goose, which died during the encephalitis outbreak in a Hungarian goose flock, and brain samples from a goshawk, which also died from encephalitis, were used for WNV nucleic acid determination. The brain samples were homogenized in ceramic mortars by using sterile quartz sand, and the homogenates were suspended in RNase-free distilled water. Samples were stored at –80°C until nucleic acid extraction was performed. Viral RNA was extracted from 140 μL of brain homogenates by using the QIAamp viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. First, a universal JEV-group specific oligonucleotide primer pair designed on the nonstructural protein 5 (NS5) and 3´-untranslated regions (UTR) of WNV (forward primer: 5´-GARTGGATGACVACRGAAGACATGCT-3´ and reverse primer: 5´-GGGGTCTCCTCTAACCTCTAGTCCTT-3´ [21]; ) was applied on the RNA extracts in a continuous RT-PCR system employing the QIAGEN OneStep RT-PCR Kit (Qiagen). Each 25-μL reaction mixture contained 5 μL of 5× buffer (final MgCl2 concentration 2.5 mmol/L), 0.4 mmol/L of each deoxynucleoside triphosphate, 10 U RNasin RNase Inhibitor (Promega, Madison, WI, USA), 20 pmol of the genomic and reverse primers, 1 μL enzyme mix (containing Omniscript and Sensiscript Reverse Transcriptases and HotStarTaq DNA polymerase) and 2.5 μL template RNA. Reverse transcription was carried out at 50°C for 30 min, followed by a denaturation step at 95°C for 15 min. Thereafter, the cDNA was amplified in 40 cycles of heat denaturation at 94°C for 40 s, primer annealing at 57°C for 50 s, and DNA extension at 72°C for 1 min, and the reaction was completed by a final extension for 7 min at 72°C. Reactions were performed in a Perkin-Elmer GeneAmp PCR System 2400 thermocycler (Wellesley, MA, USA) and in a Hybaid PCR Sprint thermocycler (Thermo Electron Corporation, Waltham, MA, USA). After RT-PCR, 10 μL of the amplicons was subjected to electrophoresis in a 1.2% Tris acetate-EDTA-agarose gel at 5 V/cm for 80 min. The gel was stained with ethidium bromide; bands were visualized under UV light and photographed with a Kodak DS Electrophoresis Documentation and Analysis System using the Kodak Digital Science 1D software program (Eastman Kodak Company, Rochester, NY, USA). Product sizes were determined with reference to a 100-bp DNA ladder (Promega). Where clear PCR products of the previously calculated sizes were observed, the fragments were excised from the gel, and DNA was extracted by using the QIAquick Gel Extraction Kit (Qiagen). Fluorescence-based direct sequencing was performed in both directions on PCR products. Sequencing of PCR products was carried out with the ABI Prism Big Dye Terminator cycle sequencing ready reaction kit (Perkin-Elmer), according to the manufacturer's instructions, and an ABI Prism 310 genetic analyzer (Perkin-Elmer) automated sequencing system. Nucleotide sequences were identified by Basic Local Alignment Search Tool (BLAST, http://www.ncbi.nlm.nih.gov/blast) search against gene bank databases. Based on the sequence information obtained from the amplification products, complete WNV sequences that exhibited the highest nucleotide identities with the Hungarian genotypes were selected from the GenBank database to design primers that amplify overlapping RT-PCR products covering the entire genome of the strains. Oligonucleotide primers were designed with the help of the Primer Designer 4 for Windows 95 (Scientific and Educational Software, Version 4.10; Microsoft, Redmond, WA, USA) and were synthesized by GibcoBRL Life Technologies, Ltd. (Paisley, Scotland, UK). Detailed information on all primers is in the Tables A1 and A2. PCR amplification products were directly sequenced in both directions; the sequences were compiled and aligned to complete genome sequences of selected representatives of WNV lineages 1a, 1b, 2, and putative lineages 3 and 4 (listed in Table). Phylogenetic analysis was performed by using the modified neighbor-joining method (ClustalX [22]; ), and trees were constructed to demonstrate the relationship between the Hungarian WNVs and other WNV strains (Figure). Figure Phylogenetic tree based on the complete nucleotide sequences of selected West Nile virus strains demonstrating the genetic relatedness of these strains (abbreviations are listed in Table). Boxes indicate different lineages and clades. The Hungarian strains reported in this article are highlighted with gray background). RabV, Rabensburg virus; JEV, Japanese encephalitis virus. Scale bar depicts degree of relatedness. Table West Nile virus strains included in the phylogenetic analysis Name Code Accession no. Isolation Year Host Origin Lineage, clade WNV HNY1999 NY99a AF202541 1999 Human New York, USA 1a WNV NY99flamingo38299 NY99b AF196835 1999 Flamingo New York, USA 1a WNV IS98STD Is98 AF481864 1998 Stork Israel 1a WNV goose-Hungary/03 Hu03 DQ118127 2003 Goose Hungary 1a WNV Italy1998Equine It98 AF404757 1998 Horse Italy 1a WNV RO9750 Ro96 AF260969 1996 Culex pipiens Romania 1a WNV VLG4 Rus99a AF317203 1999 Human Volgograd, Russia 1a WNV LEIV-Vlg99-27889 Rus99b AY277252 1999 Human Volgograd, Russia 1a WNV PaH001 Tu97 AY268133 1997 Human Tunisia 1a WNV PaAn001 Fr00 AY268132 2000 Horse France 1a WNV Eg 101 Eg51 AF260968 1951 Human Egypt 1a WNV Chin-01 Chin01 AY490240 1950s ? Russia 1a WNV Kunjin MRM61C Kunjin D00246 1960 Cx. annulirostris Australia 1b WNV Sarafend Sarafend AY688948 Laboratory strain 2 WNV B956 (WNFCG) Ug37 NC_001563 1937 Human Uganda 2 WNV goshawk-Hungary/04 Hu04 DQ116961 2004 Goshawk Hungary 2 Rabensburg virus (97-103) RabV AY765264 1997 Cx. pipiens Czech R. 3? WNV LEIV-Krnd88-190 Rus98 AY277251 1998 Dermacentor marginatus Caucasus, Russia (Georgia?) 4? The nucleotide sequences of the Hungarian WNV strains goose-Hungary/03 (Hu03) and goshawk-Hungary/04 (Hu04) were submitted to the GenBank database. They are available under accession numbers DQ118127 and DQ116961, respectively. Results In this study, the complete genome sequences of WNV strains derived from a 6-week-old goose, which died in 2003 during an outbreak of encephalitis in a Hungarian goose flock (strain goose-Hungary/03), and from a goshawk, which also died from encephalitis in the same region 1 year later (strain goshawk-Hungary/04), were determined, aligned, and phylogenetically analyzed. The genome of the goose-Hungary/03 strain is composed of 10,969 nucleotides (nt) and contains 1 open reading frame between nucleotide positions 97 and 10,398, coding for a 3,433 amino acid (aa)–long putative polyprotein precursor. The complete genomic sequence of the virus was subjected to a BLAST search against gene bank databases. The highest identity rates (98% at the nucleotide and 99% at the amino acid level) were found with WNV strains isolated in 1998 in Israel and in 1999 in the United States. In addition, phylogenetic analysis was performed to indicate the relationships between the Hungarian goose–derived WNV strain and selected representatives of WNV clades and clusters. The resulting phylogenetic tree (Figure) confirmed the results of the BLAST search, i.e., the Hungarian goose–derived WNV strain is clustering close to the previously mentioned WNV strains isolated in the United States and Israel, which belong to lineage 1a of WNV. Other European WNV strains (isolated in Italy, France, and Romania) are more distant to the Hungarian strain; they form a separate cluster consisting of a Romanian/Russian and a French/Italian subcluster. The complete nucleotide sequence of the goshawk-Hungary/04 WNV strain is composed of 11,028 nt and contains 1 open reading frame between nucleotide positions 97 and 10,401, coding for a 3,434-aa putative polyprotein precursor. In BLAST search, the strain showed the highest (96% nt and 99% aa) identity to the WNV prototype strain B 956. Consequently, as the phylogram also indicates (Figure), this virus belongs to lineage 2 of WNV. Alignments of the available partial sequences from the E protein coding regions of other representatives of this cluster showed even higher identities (97%–98% nt and 100% aa) with WNV strains isolated in central Africa in 1972 (AnB3507, AF001563) and in 1983 (HB83P55, AF001557), respectively ( 15 ). More recently (in early August 2005), additional lethal cases of encephalitis occurred in birds of prey in the same place in which the goshawk died of West Nile encephalitis in 2004, involving up to a total of 3 goshawks and 2 sparrow hawks (A. nisus); 2 of the goshawks and 1 sparrow hawk died. Preliminary investigations detected WNV-specific nucleic acid in the brains of the birds. The partial nucleotide sequence of the 2005 virus (1,000 bp at the NS5´–3´-UTR regions) showed 99.9% identity with the goshawk-Hungary/04 strain (only 1 substitution at nucleotide position 9,376 [g→a] has been observed, which did not influence the putative amino acid sequence). Additional observation of the outbreak and investigations of the cases are in progress. Discussion The primary aim of our investigations was to show the genetic relatedness of the WNV strains detected in Hungary in the last 2 years and to estimate their clinical and epidemiologic impact. The phylogenetic analysis emphasizes the close genetic relationship of the goose-Hungary/03 strain with a WNV strain isolated in Israel in 1998 and the WNV strain introduced in New York in 1999, since the 3 WNVs form 1 single cluster within clade 1a of lineage 1. These strains caused outbreaks in birds, humans, and horses. Previous European WNV isolates exhibited lower identity values, e.g., the strain that was responsible for the Romanian outbreak(s) in 1996 and 1997 showed only 96% nt identity with the Hungarian goose-2003 strain, and in the phylogenetic tree the other European isolates form a separate cluster consisting of 2 subclusters (Figure). The earliest representatives of the Israel/USA/goose-Hungary/03 cluster were reported by Malkinson et al. ( 23 ) from ill and dead white storks (Ciconia ciconia) in Israel in 1998. These storks, however, had hatched in central Europe, and during their autumn migration southwards, strong winds had blown them off course, from their usual route to Africa, to southern Israel. Malkinson et al. suspected that these birds introduced the neurovirulent genotype of WNV to Israel from their hatching place. The wetlands of southeastern Hungary are foraging and nesting habitats for storks and many other wild bird species, and the goose farm, where the WNV outbreak occurred in 2003, is located in this region. These facts, together with the close phylogenetic relatedness of the Israeli/US/Hungarian WNV strains, strongly support the theory that storks carried the neurovirulent WNV strain from central Europe (that is, from Hungary) to Israel, which sheds new light on the introduction of WNV to New York. This virus could have originated in Israel (which is the generally accepted although not proven theory) or central Europe. In both cases, however, the virus seems to have its true origin in Europe. In a recent publication, Lvov et al. suggested that WNV could have been introduced into New York by ships traveling from Black Sea ports ( 24 ). When a WNV infection was detected in 2004 in a goshawk fledgling, which died from encephalitis in the same region of Hungary in which the outbreak in geese and humans occurred during the previous year, we anticipated a WNV strain more or less identical to the genotype detected there in 2003. The genomic sequence of this strain was not closely related to the sequence of the WNV strain detected in geese in the year before, however, but belonged to the group of central African lineage 2 WNV strains. A closely related strain from this cluster (ArB3573, AF001565, and AF458349) was identified as a neuroinvasive strain of WNV in a mouse model ( 14 ). To our knowledge, this report is the first on the emergence of a lineage 2 WNV strain outside Africa. Migratory birds that had overwintered in central Africa probably introduced this exotic strain to the wetlands of Hungary. On the other hand, as the goshawk is not a migratory species, and infection occurred in August, the African WNV strain must have already successfully adapted to local mosquito vectors. Consequently, this neurotropic, exotic WNV strain may become a resident pathogen in Europe with all the possible public health consequences. Our results indicate that the WNV strains that emerged in 2 consecutive years and caused avian deaths in Hungary are epidemiologically unrelated. Genetically distinct WNV strains are circulating simultaneously yet independently in local birds and thus most likely also in local mosquito populations within the same region. They cause sporadic cases of encephalitis and also raise the possibility of spreading to other European countries or even to other continents, as happened in 1999 with another WNV strain, which resulted in a public health catastrophe in America. In addition to the above 2 novel WNVs, we recently characterized another novel flavivirus of so far unknown human pathogenicity named Rabensburg virus, which has been isolated from Culex pipiens mosquitoes in 1997 and 1999 at the Czech Republic–Austria border, only a few hundred kilometers from the region where the Hungarian WNVs emerged. After the entire genome was sequenced, Rabensburg virus turned out to represent either a new (third) lineage of WNV or a novel flavivirus of the JEV group ( 18 ). Thus, several distinct WNV strains seem to circulate in central Europe. In 2001 another flavivirus of the JEV group, Usutu virus, which has never previously been observed outside Africa, emerged in Austria and resulted in deaths in several species of birds, especially Eurasian blackbirds (Turdus merula) ( 21 ). This virus became a resident pathogen in Austria and continues to disperse and cause deaths in blackbirds and other species of birds ( 25 , 26 ). The snowy winter and rainy spring of 2005 resulted in serious floods in the area in which the Hungarian WNV strains were identified. Since the floodplains and polders were under water, the conditions for mosquito development were ideal. The summer was also very rainy, which resulted in more floods in the region and continuous mosquito gradation. The most recent data imply that the lineage 2 WNV strain may have overwintered in Hungary, causing several clinical cases of encephalitis in Accipiter species in 2005 as well. The routine diagnostic techniques in most of the European public health and veterinary laboratories are designed to detect lineage 1 WNV strains. In a recent PCR external quality assurance multicenter test, <40% of the involved laboratories could detect lineage 2 WNV strains (Matthias Niedrig, pers. comm.). Therefore, a major goal of this article is to increase the scientific and public awareness of this potential public health threat for Europe and, perhaps, America. Furthermore, comprehensive investigations on the occurrence, ecology, and epidemiology of the different WNV strains circulating in central Europe, as well as the development of monitoring and surveillance programs, must be of highest priority. One may also speculate on environmental factors, such as climate change or global warming, that may have enhanced the recent emergence of viruses, which had previously been restricted to Africa, in new habitats and continents. Improved observation, reporting, and detection methods have also contributed to the apparent increasing emergence of these viruses.

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Flagellin as an adjuvant: cellular mechanisms and potential.

Steven B Mizel, John T. Bates (2010)

Flagellin is a potent activator of a broad range of cell types involved in innate and adaptive immunity. An increasing number of studies have demonstrated the effectiveness of flagellin as an adjuvant, as well as its ability to promote cytokine production by a range of innate cell types, trigger a generalized recruitment of T and B lymphocytes to secondary lymphoid sites, and activate TLR5(+)CD11c(+) cells and T lymphocytes in a manner that is distinct from cognate Ag recognition. The plasticity of flagellin has allowed for the generation of a range of flagellin-Ag fusion proteins that have proven to be effective vaccines in animal models. This review summarizes the state of our current understanding of the adjuvant effect of flagellin and addresses important areas of current and future research interest.

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West Nile virus recombinant DNA vaccine protects mouse and horse from virus challenge and expresses in vitro a noninfectious recombinant antigen that can be used in enzyme-linked immunosorbent assays.

Nadia L. Mitchell, Gwong-Jen Chang, Denis Martin … (2001)

Introduction of West Nile (WN) virus into the United States in 1999 created major human and animal health concerns. Currently, no human or veterinary vaccine is available to prevent WN viral infection, and mosquito control is the only practical strategy to combat the spread of disease. Starting with a previously designed eukaryotic expression vector, we constructed a recombinant plasmid (pCBWN) that expressed the WN virus prM and E proteins. A single intramuscular injection of pCBWN DNA induced protective immunity, preventing WN virus infection in mice and horses. Recombinant plasmid-transformed COS-1 cells expressed and secreted high levels of WN virus prM and E proteins into the culture medium. The medium was treated with polyethylene glycol to concentrate proteins. The resultant, containing high-titered recombinant WN virus antigen, proved to be an excellent alternative to the more traditional suckling-mouse brain WN virus antigen used in the immunoglobulin M (IgM) antibody-capture and indirect IgG enzyme-linked immunosorbent assays. This recombinant antigen has great potential to become the antigen of choice and will facilitate the standardization of reagents and implementation of WN virus surveillance in the United States and elsewhere.

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Matthias Johannes Schnell: Role: Editor

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Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS ONE

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, CA USA )

ISSN (Electronic): 1932-6203

Publication date (Electronic): 22 February 2016

Publication date Collection: 2016

Volume: 11

Issue: 2

Electronic Location Identifier: e0149610

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[1 ]Infectious Disease Research Institute, 1616 Eastlake Ave E., Seattle, WA 98103, United States of America

[2 ]Colorado State University Department of Biomedical Sciences, Foothills Campus, Fort Collins, CO 80523, United States of America

[3 ]Hawaii Biotech Inc. 99-193 Aiea Heights Drive, Aiea, Hawaii 96701, United States of America

Thomas Jefferson University, UNITED STATES

Author notes

Competing Interests: David Clements, Elliot Parks, and Timothy Martyak are employees of Hawaii Biotech, a privately held biotechnology company developing vaccines. They have provided the WN-80E antigen used and described in this work. Neal Van Hoeven, David Clements, and Elliot Parks are inventors on a patent that is expected to be filed on work described in this manuscript during 2016. There are no further patents, products in development, or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Conceived and designed the experiments: NVH RC RAB. Performed the experiments: SWJ GIN AB CF RAB NVH. Analyzed the data: NVH SWJ GIN AB RAB. Contributed reagents/materials/analysis tools: CF DEC TM DEP. Wrote the paper: NVH SWJ GIN SB SR RC.

* E-mail: neal.vanhoeven@ 123456idri.org

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Publisher ID: PONE-D-15-47361

DOI: 10.1371/journal.pone.0149610

PMC ID: 4762984

PubMed ID: 26901122

SO-VID: 942e7c7b-a320-41a1-9d09-1f85a120d00a

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This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Date received : 2 November 2015

Date accepted : 2 February 2016

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Figures: 6, Tables: 1, Pages: 20

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This research was supported with funding from the Bill and Melinda Gates Foundation, under grant OPP1055855, and with unrestricted IDRI funds. The external funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Hawaii Biotech Inc. provided support in the form of salaries for authors DEC, TM, and DEP, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the 'author contributions' section.

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A Novel Synthetic TLR-4 Agonist Adjuvant Increases the Protective Response to a Clinical-Stage West Nile Virus Vaccine Antigen in Multiple Formulations

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Lineage 1 and 2 Strains of Encephalitic West Nile Virus, Central Europe

Flagellin as an adjuvant: cellular mechanisms and potential.

West Nile virus recombinant DNA vaccine protects mouse and horse from virus challenge and expresses in vitro a noninfectious recombinant antigen that can be used in enzyme-linked immunosorbent assays.

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