- Neurospora crassa is being investigated as a system for producing subunit vaccines through the secretion and purification of influenza hemagglutinin (HA) and neuraminidase (NA) antigens. - HA and NA from influenza A/New Caledonia/20/1999 were expressed successfully in N. crassa with HA exhibiting hemagglutination activity. HA was purified using nickel affinity chromatography. - The rapid growth and simple culture of N. crassa allows for lower cost, higher yield vaccine production than traditional egg- or mammalian cell-based systems. This could help address vaccine supply issues.

1. Vaccine production in Neurospora crassa
Silke Allgaier1
, Rebecca D. Taylor1
, Yuliya Brudnaya, David J. Jacobson,
Edward Cambareri, W. Dorsey Stuart*
Neugenesis Corporation, 849 Mitten Road, Suite 102, Burlingame, CA, USA
Received 2 February 2009; accepted 2 February 2009
Abstract
We have chosen to use the filamentous fungus Neurospora crassa to produce subunit vaccines. Here we describe the production and
purification of Influenza hemagglutinin and neuraminidase antigens in N. crassa. The N. crassa system used by Neugenesis offers many
advantages over other systems for production of recombinant protein. In contrast to mammalian cell culture, N. crassa can be grown in a rapid
and economic manner, generating large amounts of recombinant protein in simple, defined medium. Vaccines, therefore, can be produced more
rapidly and at lower cost than conventional cell culture or egg-based systems. This has important applications to tailoring the seasonal vaccine
supply and responding to new pandemics.
Ó 2009 Published by Elsevier Ltd on behalf of The International Association for Biologicals.
Keywords: Neurospora crassa; Filamentous fungi; Heterologous protein production; Influenza vaccine; Hemagglutinin; Neuraminidase
1. Introduction
For decades filamentous fungi have been used successfully
for the production and secretion of recombinant proteins [1].
Trichoderma reesei and a variety of Aspergillus species are the
primary fungal species utilized for recombinant protein
expression [2]. Optimization of wild-type strains has resulted
in impressive improvements in protein yield. In commercial
settings optimized fermentation processes have resulted in
yields of >30 g/L of recombinant protein [3]. Although not
a common industrial organism, Neurospora crassa is our
choice of a fungal system for production of recombinant
protein, as it is well characterized, both genetically and bio-
chemically [4]. An additional advantage is that the N. crassa
genome has been fully sequenced and a community effort is
underway to knock-out every open reading frame in the
genome [5]. All the knock-outs are publicly available via the
Fungal Genetics Stock Center (www.fgsc.net).
The first vaccine candidate we have chosen to develop is
directed against a seasonal H1N1 variant of influenza, (A/New
Caledonia/20/99). The influenza virion consists of a lipid
bilayer envelope derived from the host cell, viral proteins and
eight minus strand RNAs that encode the genome of the virus.
The surface glycoproteins of the influenza virion compose the
dominant antigenic targets of the immune system; the main
proteins in the membrane are hemagglutinin (HA), which
facilitates entry of the virion into human cells, and neuramin-
idase (NA), which both prevents aggregation of the virion
within the host cells and facilitates virion infection from cell to
cell. A key structural protein, M1, is located within the virion
envelope and has been found to facilitate virion assembly [6].
Currently, influenza vaccines are produced either in co-infec-
ted, embryonated chicken eggs [7] or using cell culture based
production in insect cell lines or mammalian cell lines [8].
The use of N. crassa to produce antigenic recombinant
protein will decrease both the time and cost for vaccine
production. As proof of concept, we show secretion and partial
purification of active recombinant HA and NA from N. crassa.
In addition, Neugenesis heterokaryon technology (US Patents
5643745, 5683899, 6268140) allows the production of flexible
combinations of subunit-multimer proteins like mAbs or
* Corresponding author. Fax: þ1 650 259 9435.
E-mail address: dstuart@neugenesis.com (W.D. Stuart).
1
First two authors contributed equally to this work.
1045-1056/09/$36.00 Ó 2009 Published by Elsevier Ltd on behalf of The International Association for Biologicals.
doi:10.1016/j.biologicals.2009.02.006
Available online at www.sciencedirect.com
Biologicals 37 (2009) 128e132
www.elsevier.com/locate/biologicals

2. multivalent vaccine mixtures. Here, we demonstrate this
technology by using it to produce virus-like particles (VLPs)
that display viral glycoproteins on the surface of a secreted
membrane-bound particle which facilitates the purification of
subunit vaccines that can be used for vaccination.
2. Materials and methods
2.1. Expression plasmid construction
Full-length hemagglutinin (HA, AAP34324) and neuramin-
idase (NA, ABF21328.1) sequences from Influenza A (A/New
Caledonia/20/1999/H1N1) were obtained from the Flu database
of the Influenza Virus Resource at NCBI. A truncated version of
the HA amino-acid sequence was designed replacing the native
signal peptide with a fungal leader from the Cel12A gene of T.
reesei (MKFLQVLPALIPAALAQ). In addition, the C-terminus
of this sequence was deleted after residue isoleucine 516 of the
native sequence, creating a soluble ectodomain version of the
HA. In contrast, the NA amino-acid sequence was maintained as
the full-length version of the gene, which contained in addition
to the native sequence, unique BglII and XbaI sites on the 50
and
30
ends, respectively, for ease of cloning into the final expression
vector (Fig. 1).
The sequences were then optimized for expression with N.
crassa codon usage tables, using Gene Designer, version
1.0.7.1. The codon-optimized genes were scanned and adjusted
for newly created splice-donor and acceptor sequences and
transcriptional terminator sites, and synthesised by DNA2.0
(Redwood City, CA). The synthetic genes were then subcloned
into a vector containing the promoter and terminator sequences
of the Aspergillus niger glucoamylase (GlaA), creating the
plasmids pHDHA1, and pHDNAnc. Plasmid pHDHA1 was
then modified using PCR to generate a version of HA with
a histidine tag (9 Â His) at the C-terminus of the truncated HA,
resulting in plasmid pHDHAH9. Both vector constructs con-
tained a portion of the his-3 gene which facilitates targeting of
the construct to the his-3 locus [9].
2.2. Strain constructions and cell growth
The HA (pHDHAH9) and NA constructs (pHDNAnc) were
transformed into host strains with a mutation in the native
his-3 locus. Transformants were screened by selection for
histidine prototrophy, forcing recombination to occur at the
his-3 locus. This was confirmed by Southern blot (data not
shown), creating the strains expressing HA and NA. General
growth and handling of N. crassa strains was carried out as
previously described [10]. Cultures used to express vaccine
antigens were grown for three days in shake flasks in modified
Neurospora media. Culture supernatant from shake flasks was
harvested through Miracloth (Calbiochem, San Diego, CA)
and filtered through 0.45 mm PES membrane (Nalgene,
Thermo Fisher Scientific, Waltham, MA). Total protein
concentration was determined with a Coomassie-based dye
binding assay (Pierce, Thermo Fisher Scientific, Waltham,
MA). For analysis 20 ml of culture supernatant was run on
SDS polyacrylamide gels (Bio-Rad, Hercules, CA).
2.3. Red blood cell hemagglutination assay
Cultures were tested for hemagglutinin activity by adding
filtered N. crassa culture supernatant, serial diluted in a round
bottom 96 well plate, to an equal amount of a 0.6% solution
of washed Sheep or Human red blood cells (Rockland,
Gilbertsville, PA) in 1Â PBS. Plates were then incubated at
room temperature for 30 mine1 h (Fig. 2A). Samples were
compared to a known amount of purified HA standard
(Protein Sciences, Meriden, CT).
2.4. HA isolation
Supernatant from three-day-old shake flask cultures was
precipitated with the addition of 454.4 g of ammonium sulphate
at 4
C. The sample was centrifuged at 4000 g for 30 min.
Pelleted protein was resuspended in His-binding buffer (50 mM,
pH 8 phosphate buffer, 150 mM NaCl) plus 10 mM imidazole
(Sigma, St. Louis, MO) and dialyzed (10e14 kDa MWCO)
against His-binding buffer and 200 mM PMSF (Sigma, St. Louis,
MO) overnight at 4
C. Dialyzed protein was bound to Ni-NTA
agarose (Qiagen, Valencia, CA) for 1 h at room temperature. The
sample was applied to three different columns and subjected to
three wash conditions. Wash buffer consisted of His-binding
buffer containing either 10 mM, 20 mM, or 30 mM imidazole.
Bound protein was eluted in His-binding buffer with 1 ml of
250 mM imidazole. Eluted samples (20 ml) were subject to SDS-
PAGE and the gel was silver stained (Silver Xpress, Invitrogen,
Carlsbad, CA). Blue-native gel electrophoresis was performed as
per suppliers’ recommendations (NativePAGEÔ, Invitrogen,
Carlsbad, CA) with the modification that 1% 3-[(3-Chol-
amidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)
was used in the solubilization buffer in place of 1% Digitonin.
The equivalent of 600 ml of the 9 Â His-tagged HA samples
purified using Ni-NTA and 600 ng of standard HA (Protein
Sciences, Meriden, CT) were run on blue-native gels. Protein was
transferred to nitrocellulose membrane using standard techniques
Fig. 1. Plasmids used for expression of heterologous Influenza protein.
Plasmid constructs were designed to integrate histidine tagged hemagglutinin
(HA þ 9 Â His) and neuraminidase (NA) from Influenza A (A/New Caledonia/
20/1999/H1N1) at the his-3 locus of Neurospora crassa. Both plasmids are
driven by the Aspergillus niger glucoamylase promoter as this promoter has
been shown to be a strong constitutive promoter in N. crassa. (A) The
hemagglutinin gene is fused at the N-terminus to the first 17 amino acids of
Cel12A from A. niger which constitutes the signal sequence of the protein
directing HA to the ER. A 9Â histidine tag was added to the construct by PCR.
(B) The neuraminidase gene in pHDNAnc contains the native neuraminidase
signal sequence.
129S. Allgaier et al. / Biologicals 37 (2009) 128e132

3. [11] and visualized using a primary mAb directed against H1 HA
(BioDesign, Saco, ME), followed by a secondary anti-mouse IgG
antibody conjugated with alkaline phosphatase (Rockland, Gil-
bertsville, PA). Color was developed on membranes using BCP/
NBT reagent (Pierce, Thermo Fisher Scientific, Waltham, MA).
2.5. NA expression and isolation
NA was expressed using a N. crassa heterokaryon fusion of
strains expressing either the M1 protein or the Neuraminidase
N1 from the Influenza A virus (A/New Caledonia/20/1999).
Supernatant from three-day-old shake flask cultures was
purified according to Bucher [12] with the following modifi-
cations: to crude extracts, 2% Triton X-100 was added and
stirred for 1 h before adjusting the pH to 5.5. Adjusted crude
extracts were loaded directly on 5 ml of a pre-equilibrated
N-(p-Aminophenyl)oxamic acideagarose column (Sigma, St.
Louis, MO). Two washes were performed using equilibration
buffer (50 mM NaAcetate, 2 mM CaCl2, 0.1% Triton X-100)
and 50 mM TriseHCl, 2 mM CaCl2, 0.1% Triton X-100.
Bound protein was eluted using 25 mM diethanolamine, 2 mM
CaCl2, 0.1% Triton X-100. All purifications were done on
Fig. 2. Recombinant hemagglutinin expressed in Neurospora crassa. (A) Red blood cell hemagglutination assay. Media containing active HA is mixed with 0.6%
washed Human red blood cells. Diffuse color indicates presence of active HA, as HA binds red blood cells and forms a reticular net and thus prevents pooling of
red bloods cells at the well bottom. Dark red spots indicate the absence of active HA as red blood cells pool in the well bottoms. (B) Hemagglutination activity of
culture supernatant expressing HA. HA, purified commercially available recombinant HA (Protein Sciences) starting at a concentration of 1 mg/ml, H1-3,
Neurospora strains expressing HA, Ho, untransformed host strain. (C) Ni-NTA purification of HA with nine histidine residues at the C-terminus of the protein.
Purification was split into three and washed with differing concentrations of imidazole, 10 mM (10), 20 mM (20) or 30 mM (30). 50 ng of purified commercially
available HA standard (Std) was loaded on gel as well as a fraction of the column flow through. (D) Western blot of blue-native gel of commercially available HA
standard (HA std.), 600 ng, and 9 Â His-tagged HA (HAeNc) purified from culture supernatant. Membrane was decorated with anti-HA antibody (Bio Design).
130 S. Allgaier et al. / Biologicals 37 (2009) 128e132

4. a BioCAD/RPM system (Perceptive Biosystems, Framingham,
MA). Fractions were assayed for neuraminidase activity using
the AmplexÒ
Red neuraminidase (Sialidase) Assay Kit
(Invitrogen, Carlsbad, CA).
3. Results
As a first step towards the creation of a subunit vaccine,
sequences of HA and NA from seasonal influenza strain
(A/New Caledonia/20/1999/H1N1) were obtained and cloned
as described in materials and methods (Fig. 1). The optimized
NA gene was placed into the plasmid intact with its endoge-
nous signal sequence, directing the protein to the plasma
membrane (Fig. 1A). The expressed version of the HA protein
was modified. First, the transmembrane and cytoplasmic
domains were removed from the protein to allow soluble
secretion directly into the medium. Second, we replaced the
native signal sequence of HA with one from the well-
expressed CEL12A (EGIII) protein from T. reesei (Fig. 1B) to
ensure efficient expression and secretion. Third, nine histidine
residues (9 Â His) were placed at the C-terminus of the protein
as a tag for simplified downstream purification of the protein
using standard Ni-NTA protein purification techniques. The
resulting plasmids were transformed into N. crassa. Trans-
formants were grown for three days and assayed to determine
the best expressing strains for further work. To determine if
the expressed HA was fully functional a hemagglutination
assay was performed (Fig. 2A). The HA expressing trans-
formants demonstrated red blood cell agglutination activity
(Fig. 2B, H1eH3) similar to that of the commercially avail-
able HA. No red blood cell agglutination activity could be
demonstrated in the non-transformed host (Fig. 2B, Ho). Thus
HA produced by N. crassa was active and could associate into
multimeric complexes, as this is required for agglutination
activity.
Large-scale cultures of the best expressing strains were
grown up for protein purification. Culture supernatant was
precipitated with ammonium sulphate to exchange culture
media with a buffer that was compatible with the Ni-NTA
purification and to eliminate contaminating host proteins.
Precipitated protein was resuspended in His-isolation buffer
and dialyzed against the same buffer overnight. The dialyzed
protein was bound to Ni-NTA sepharose. The sample was split
into three and each of the three portions was subjected to wash
conditions with varying amounts of imidazole. The inclusion
of imidazole in the wash buffer resulted in a clean HA pre-
paration with little background protein contamination.
Following elution of bound protein, a small amount of each
fraction were subjected to SDS-PAGE (Fig. 2C) and blue-
native PAGE (Fig. 2D). As predicted, the protein purified from
the Ni-NTA column was slightly smaller than the native HA
standard due to the deletion of the transmembrane domain
(Fig. 2C, lane 4e6). In addition, blue-native PAGE showed
that HA purified from N. crassa culture supernatant showed
the same folding pattern as the native HA standard (Fig. 2D).
Taken together these data show that HA was produced by
N. crassa in an active conformation and that the protein had
native or near native protein folding when compared to
a commercially available HA used in vaccine preparation
(FlublØk, Protein Sciences, Meriden, CT).
We took an alternative approach for the expression of NA
to ensure that this Type II membrane protein would assemble
into antigenic multimers, but maintain a simplified purification
from culture medium. This can be achieved by using secreted
virus-like particles (VLPs) displaying a membrane glycopro-
tein for purification. It is well established that co-expression of
Influenza membrane-bound antigens with the M1 protein
results in lipid-rafting and budding of virus-like particles in
heterologous systems [13]. We wanted to determine if
Neurospora was also capable of secreting high-molecular
weight particles containing NA. To do this, the pHDNAnc
expression plasmid was transformed into N. crassa and the
resulting strains were tested for NA expression. Neuramini-
dase assays were performed on heterokaryons of NA trans-
formants that were fused to a strain bearing the M1 expression
construct. Culture supernatants were tested for neuraminidase
activity and all strains showed a high activity compared to an
untransformed host strain (Fig. 3A). Size exclusion chroma-
tography using a Sephadex G100 column was performed on
media from heterokaryon cultures, and the exclusion volume
was demonstrated to contain NA by Western blot (data not
shown), indicating that this protein is associated with high-
molecular weight species (presumably VLPs), therefore
allowing a simplified approach for purification.
Large-scale cultures of the best expressing strains were
grown up to produce a sufficient amount of VLPs for subse-
quent vaccine purification. To disrupt the virus-like particles
and stabilize NA, Triton X-100 was added to the culture prior
to purification. Samples were loaded on N-(p-Amino-
phenyl)oxamic acideagarose, a column with affinity for sia-
lidases and neuraminidases. Following elution, a small amount
of each fraction was subjected to SDS-PAGE (Fig. 3B) and
tested for neuraminidase activity (Fig. 3C). The resulting
fractions showed the monomeric NA at the predicted size of
w50 kDa and the homotetramer at a size w200 kDa (Fig. 3B)
and results were confirmed by Western blot (data not shown).
Unlike the HA, fractions containing NA showed a higher
degree of host protein contamination, requiring additional
purification steps.
4. Discussion
In this study Neugenesis has developed techniques to
produce a variety of different antigens in the filamentous fungus
N. crassa. We have shown that HA and NA produced by
N. crassa are functional and, in the case of HA, show similar
activity when compared to commercially available HA protein.
These results also demonstrate that high-molecular weight
particles containing NA can be produced in a heterokaryon
expression system and can be used to facilitate purification of
membrane proteins.
The Neugenesis system provides several advantages over
systems currently used for vaccine production. First, vaccines
can be produced in N. crassa rapidly and at lower cost than
131S. Allgaier et al. / Biologicals 37 (2009) 128e132

5. conventional cell culture or egg-based systems. Discrete event
simulation modelling performed by BioPharm Services
(Chesham, UK) demonstrates that cost of goods can be
$0.10/dose, at titer levels of 100 mg/L using 200 L
fermentors. Lead times for a vaccine campaign are also
significantly reduced, potentially as short as 12 weeks,
allowing more rapid response to new surveillance data. We
have extended this technology to produce multivalent vaccines
by combining strains producing various HA and NA subtypes.
By creating a heterokaryon with a second strain producing the
M1 protein, the resulting strain is able to produce non-infec-
tious virus-like particles lacking viral components necessary
for pathogenicity, while retaining full antigenicity. In other
systems, these VLP antigens are proving to be ideal candidates
for vaccine formulation [14]. The ease with which different
strains of Neurospora may be fused to form heterokaryons to
express a mixture of co-expressed antigens can potentially be
developed into a powerful new approach to counter antigenic
drift and shift of Influenza populations. This can be directly
applied to tailoring the seasonal vaccine supply to create
vaccines that more effectively immunize populations at risk
for infection. Finally, this flexible combinatorial expression
technology can be applied to other pathogens that are similarly
diverse or rapidly changing in response to infection in human
populations.
Acknowledgements
Supported in part by funds from the US Defense Advanced
Research Projects Agency under its Accelerated Manufacture
of Pharmaceuticals Program. We would like to thank K. Anoruo
and K. Takeoka for their expert technical assistance.
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Fig. 3. Recombinant neuraminidase expressed in Neurospora crassa (A)
Neuraminidase activity from neuraminidase expressing cultures. Top well
starts at a 4-fold dilution of culture media. mU, milli-units of activity, N1-3,
neuraminidase expressing cultures, Ho, untransformed host strain, C, Neur-
aminidase from Clostridium perfringens (B) Neuraminidase purification from
culture supernatant expressing neuraminidase activity. Shown is a non-
reducing 4e12% BiseTris gel, the estimated size of the tetramer, w200 kDa,
and the size of the monomer, w50 kDa, are marked with arrows. M, Marker,
Fr., Fraction, Mo, Monomer, T, Tetramer. (C) Neuraminidase activity from gel
fractions shown in B. Fr., Fraction.
132 S. Allgaier et al. / Biologicals 37 (2009) 128e132