extfieldsofstudy: Mathematics Medicine

text:
The spreading frontiers of avian-human influenza described by the free boundary

In this paper, a reaction-diffusion system is proposed to investigate avian-human influenza. Two free boundaries are introduced to describe the spreading frontiers of the avian influenza. The basic reproduction numbers r 0F (t) and R 0F(t) are defined for the bird with the avian influenza and for the human with the mutant avian influenza of the free boundary problem, respectively. Properties of these two time-dependent basic reproduction numbers are obtained. Sufficient conditions both for spreading and for vanishing of the avian influenza are given. It is shown that if r 0F (0) < 1 and the initial number of the infected birds is small, the avian influenza vanishes in the bird world. Furthermore, if r 0F (0) < 1 and R 0F(0) < 1, the avian influenza vanishes in the bird and human worlds. In the case that r 0F (0) < 1 and R 0F(0) > 1, spreading of the mutant avian influenza in the human world is possible. It is also shown that if r 0F (t 0) ⩾ 1 for any t 0 ⩾ 0, the avian influenza spreads in the bird world.

Introduction
In history, epidemic diseases swept through countries and made people suffer from big disasters. The plague flourished four times in Europe, and it killed more than 15 percent population in the affected areas each time [2,6]. In 6th century, the first outbreak caused about half of European people dead; the second outbreak occurred in 1346-1350 and the rate of the mortality was 33.33%, the third outbreak killed 1 6 population of London in 1665-1666. The last happened in 1720-1722, the death rate was very high, which resulted in half population dead in Marseilles [2]. The prevalent of the disease usually leads the population declined sharply. Beside the Black Death, there are many contagious diseases, such as smallpox, malaria, yellow fever and so on.
At present, about half of the world's population is threatened with different infectious diseases [5]. Today the WHO (World Health Organization) claims that the infectious diseases are still hostile in the human world. From now on, people should be cautious about the diseases and scientists should contribute themselves to eradicate epidemic diseases. Recently, great attention has been paid to study the contagious diseases. In early 20th century, mathematical models have been constructed to study the dynamics of H(t) = β 2 SH + εB − (μ + α + γ)H, R(t) = γH − μR, (1.1) where the first two equations form the SI model, which describes the bird system, no recovered bird exists since the highly pathogenic viruses have high death rates. X and Y are the population of the susceptible birds and the infective birds, respectively. The last four equations are SIR model for humans, S, B, H and R denote, respectively, the population of the susceptible humans, infected with avian influenza, infected with mutant avian influenza and recovered humans from mutant avian influenza. The reproductive number r 0 = cω b(b+m) in the bird system and the reproductive number R 0 = β2λ μ(μ+α+γ) in the human system are defined. The authors showed that when r 0 1 and R 0 > 1, the human-endemic equilibrium (i.e., the boundary equilibrium) E h := (X 0 , 0, S, 0, H, R) is global asymptotically stable, while if r 0 > 1, the full-endemic equilibrium (i.e., the interior equilibrium) is global asymptotically stable. In other words, mutant avian influenza spreads in the human world in the above two cases.
Considering spatial spreading and time delay, Kim et al. [12] introduced diffusion terms and extended the system (1.1) to the following reaction diffusion system: K(x, y, t − s)H(s, y)dsdy + εB − (μ + α + γ)H, for t > 0, x ∈ Ω. The system (1.2) with a no-flow boundary condition is discussed, and the basic reproductive numbers r 0 and R 0 are defined the same as those in [8]. They concluded that the diseasefree equilibrium E 0 := (c/b, 0, λ/μ, 0, 0, 0) is locally asymptotically stable if r 0 < 1 and R 0 < 1; and human-endemic equilibrium E h is locally asymptotically stable if r 0 < 1 and R 0 > 1; while r 0 > 1 the full-endemic equilibrium E + is locally asymptotically stable. Furthermore, they proved the disease-free equilibrium E 0 is globally asymptotically stable when r 0 < 1 and R 0 < 1. It is known that the solution to System (1.2) with Dirichlet or Neumann boundary condition in a bounded domain is always positive for any time t > 0 no matter what the nonnegative nontrivial initial date is. It means that the avian influenza spreads to the whole area immediately even when the infectious is confined to a small part of the area at the beginning. It does not match the observed fact that the avian influenza always spreads gradually. To describe the gradual progress of avian influenza spreading, we are attempting to consider an avian-human influenza model with free boundaries, which describe the spreading frontiers of the virus. In the next section, a mathematical model of the free boundary in both the bird world and the human world is constructed first.
In Section 3, we give the local existence and uniqueness of the solution to Problem (2.4), then prove that the right free boundary x = h(t) is increasing and the left boundary x = g(t) is decreasing. Finally, we conclude that the solution to (2.4) is global and unique. Section 4 is devoted to the basic reproduction numbers r F 0 (t) for avian influenza in the bird system with free boundary and R F 0 (t) for the mutant avian influenza in the human system with free boundary. It is shown that r F 0 (t) and R F 0 (t) are strictly increasing with respect to t. Moreover, if lim t→∞ (h(t)−g(t)) = ∞, then lim t→∞ r F 0 (t) = r 0 and lim t→∞ R F is the basic reproduction number for avian influenza in the bird system (4.1), and R 0 (= βm d h +γ h ) is the basic reproduction number for the mutant avian influenza in the human system (4.1).
Sections 5-7 deal with the asymptotic behaviors of the free boundaries and the solution. In Section 5, we first prove that the free boundaries x = h(t) and x = g(t) are either finite or infinite at the same time. Then we show that if ,h(t)]) = 0, i.e., the avian influenza vanishes in the bird world. Finally by constructing suitable upper solution to problem (2.4), we give the sufficient conditions for the avian influenza to vanish in the bird and human worlds, i.e., if r F 0 (0) < 1, R F 0 (0) < 1 and the initial infected data are small enough, then h ∞ − g ∞ < ∞ and the solution decays gradually toward zero.
In Section 6, we investigate the case that r F 0 (0) < 1 and .
In this situation, the mutant avian influenza transmits in the human. In Section 7, we show that if r F 0 (t 0 ) 1 for any t 0 0, the avian influenza spreads in the bird world. Furthermore, we demonstrate that if r F 0 (0) < 1 < r 0 and B i0 (x) is big enough, then In other words, the avian influenza can spread in the whole area even that r F 0 (0) < 1. A short discussion is given in Section 8.

Model formulation
We consider a general avian-human model and classify the birds and humans as the following categories: • Susceptible birds B s (t), infected birds B i (t) with avian influenza; • Susceptible humans H s (t), infected humans H a (t) with avian influenza, infected humans H m (t) with mutant avian influenza and recovered ones H r (t) from mutant avian influenza.
Here, we assume that all birds infected with avian influenza are dead or remain infected and can be never recovered. Then the bird system can be described by SI model. But the human infected with mutant avian influenza can be cured, then the person has immunity against avian influenza virus in all his/her life. So we can use the SIR model to represent the human world. If we do not consider the spatial spreading of the virus, an ODE model reads where G(B s , B i ) is the per capita reproduction rate of the adult birds which can be taken as r b (1− Bs+Bi K b ), or simply a constant r b ; d b and d h are the death rates with the bird and the human; β a , β b and β m denote the contact rates of the virus from infected bird with avian influenza to human, infected bird to bird and infected human with mutant avian influenza to human, respectively; r h is the birth rate of human; γ h denotes the recover rate of human from the mutant avian influenza virus; ε h is the mutant rate with the avian influenza virus. N b and N h denote the total numbers of the bird and the human, respectively.
In general, the individual disperses randomly in the habitat. Therefore, we consider not only the individual's activity in temporal dimension, but also the distribution of the individual in the spatial and the dynamic characteristic of the avian influenza. To describe the diffusion of the disease, we introduce the spatial diffusion terms. We start with one-dimensional case: −∞ < x < ∞, thus an extended version of the avian-human model can be described by for −∞ < x < ∞ and t > 0, where D b and D h are positive diffusion coefficients for the birds and humans, respectively. For simplicity, we take G(B s , B i ) = r b , and let In other words, we assume the total number of birds and that of humans remain constant.
This research is devoted to the transmission of avian influenza. Despite the infected is limited in a small district, the avian influenza can spread in the whole habitat immediately. Obviously, it cannot be used to describe the real spreading of the avian influenza virus. We now use the free boundary to describe the spreading frontier of the disease. Assume that the birds and humans migrate in the whole habitat (−∞, ∞), and some birds are infected in g(t) < x < h(t), there is only the susceptible birds or humans on the rest part. The right spreading frontier is represented by the free boundary x = h(t). Assume that h(t) grows at a rate that is proportional to the population gradient at the frontier [7,11,17,18,28]. Then the conditions on the right frontier (free boundary) is Similarly, the conditions on the left frontier (free boundary) is In such a case, we have the problem for where x = g(t) and x = h(t) are the moving left and right boundaries to be determined, h 0 and μ are positive constants, and the initial functions B i0 , H a0 , H m0 and H r0 are nonnegative and satisfy Noting that the first equation of the system (2.4) is independent of the last three equations, we can consider the bird system as the following: In the rest part, we will consider (2.4) and (2.6), then give the properties of the solution and the free boundaries.

Existence and uniqueness
In this section, a contraction mapping argument is used to show the local existence and uniqueness of the solution to (2.4). Then we use standard methods (such as Hopf Lemma, the maximum principle) to derive the estimates of the solution and present the global existence and uniqueness of the solution. First, the following local existence result can be proved by the contraction mapping argument as in [4].
According to (2.5) and the equation of By using maximum principle, it is easy to see that 0 To explain that the local solution to (2.4) can be extended to all t > 0, we need the following lemma to show the free boundaries for problem (2.4) are strictly monotonous.
Using the Hopf lemma to the equation of B i at x = g(t) and x = h(t) yields that Hence, g (t) < 0 and h (t) > 0 for t ∈ (0, T 0 ) by using the free boundary conditions in (2.4). It remains to show that −g (t), h (t) C 2 for t ∈ (0, T 0 ) and some C 2 independent of T 0 . Inspired by [7], define For any t ∈ (0, ∞), the solution to (2.4) exists and is unique.

Proof.
It follows from the uniqueness of the solution that there is a number T max such that [0, T max ) is the maximal time interval in which the solution exists. Now, we show that T max = ∞ by the contradiction argument.
Assume that T max < ∞. By Lemma 3.2, there exists C 2 independent of T max such that We now fix δ 0 ∈ (0, T max ) and M > T max . By standard parabolic regularity, we can find

Basic reproduction numbers
In this section, we denote the basic reproduction numbers for different systems and present their properties which will be used in the sequel. Now we study the full system (2.3). The corresponding ODE system is governed by Similarly to that in [8], the basic reproduction number r 0 of the bird and the basic reproduction number R 0 for mutant avian influenza in the human world are represented by According to [8,Theorem 3.2], if r 0 < 1 and R 0 < 1, then DFE is locally asymptotically stable; the HEE is locally asymptotically stable if r 0 < 1 and R 0 > 1, and if r 0 > 1, the FEE is locally asymptotically stable.
If the environment Ω is heterogeneous and the boundary of region Ω is hostile to the population for living (see [23]), then the corresponding system is given by Let us introduce the basic reproduction numbers for the bird r D 0 (Ω) and the mutant human R D 0 (Ω), respectively, as follows: and λ R0 be the first eigenvalue of Furthermore, its corresponding eigenfunctions ϕ λr 0 and φ λR 0 can be chosen to be positive on Ω. Obviously, λ r0 and λ R0 can be given by variational characterization,

Lemma 4.2.
The following properties hold: One can directly verify this statement (see [ where λ(Ω) is the principle eigenvalue of the following eigenvalue problem: It is well known that λ(Ω) is a strictly decreasing continuous function and that From the direct calculations, the following lemma holds.
(iv) Let B ρ be a ball with radius ρ. Then r D 0 (B ρ ) and R D 0 (B ρ ) are strictly monotonically increasing function of ρ. That is to say, By the free boundary problem (2.4), we know that the domain (g(t), h(t)) is changing with respect to t. Thus the basic reproduction number is a function with t. Now, we define the basic reproduction numbers r F 0 (t) and R F 0 (t) for the free boundary problem (2.4) by .
This conclusion follows directly from Lemmas 3.2 and 4.3.

Vanishing of the avian influenza
In this section, we will consider the vanishing of the avian influenza in the bird and in the human world. First, we show that the double free boundary frontiers x = g(t) and x = h(t) are either finite or infinite at the same time.  Suppose that (B i , H a , H m , H r ; h, g) is a solution to (2.4) defined for x ∈ [g(t), h(t)] and t ∈ [0, ∞). Then we have

Proof.
For small t > 0, we have g(t) + h(t) > −2h 0 by the continuity. Define Now, we show that T = ∞ by the contradiction argument. Assume that there exists T with 0 < T < ∞ such that Furthermore, we get Direct calculation shows that Noting that C(x, t) ∈ L ∞ and then by using the comparison principle, we obtain Applying the strong maximum principle gives that However, By the Hopf lemma, we have B x (g(T ), T ) < 0. Moreover, Then we have which is a contradiction to (5.1). Hence we have shown and g(t) + h(t) < 2h 0 for all t 0 can be proven by the same argument.
It follows from the regularity of the parabolic equation that {B n } has a subsequence {B ni } such that Moreover, B(x 0 , 0) σ 2 , and thus we can get Applying the Hopf lemma yields that there exists > 0 such that B x (h ∞ , 0) − . It follows from Theorem 3.1 and Lemma 3.2 that −g(t) and h(t) are monotonically increasing and bounded. By the standard L p theory and the Sobolev imbedding theorem (see [13]), for any 0 < α < 1, there exists a constant C which depends on α, h 0 , B i0 C 1+α ([−h0,h0]) , g ∞ and h ∞ such that On the other hand, since h(t) is bounded, we have h (t) → 0 as t → +∞. So ∂Bi ∂x (h(t n ), t n ) → 0 as n → +∞ by the free boundary condition. Noting This is a contradiction to that B x (h ∞ , 0) − . Hence, lim t→+∞ B i (·, t) C([g(t),h(t)]) = 0.
Lemma 5.2 implies the following result.
To give the sufficient conditions for the avian influenza to vanish, we first give the definition of the upper and the lower solutions to the bird system, then give the comparison principle.
is an upper solution to the bird system (2.6).

h(t) h(t), g g(t) g(t), t ∈ (0, T ],
Now, we give the sufficient condition for the avian influenza to vanish in the bird world. h0,h0]) is sufficiently small.
The following comparison principle for the full system can be obtained similarly to [7,Lemma 3.5].

Discussion
Most prior work related to the disease transmitting has focused on the understanding of the disease dynamic in the fixed domain. There is almost no work which has investigated the characteristics of disease spreading in a moving area.
In this paper, we introduce two moving boundaries, which are called free boundaries, to describe the avian influenza virus transmitting in the habitat. The dynamical behaviors of the solution have been discussed. Two basic reproduction numbers r F 0 (t) and R F 0 (t) are given for the free boundary problem (2.4) in Section 4. The infected domain (g(t), h(t)) changes with t, so the basic reproduction numbers depend on time t. It has been shown that the two reproduction numbers r F 0 (t) and R F 0 (t) are monotonically increasing with respect to t, and r F 0 (t) and R F 0 (t) approach to r 0 and R 0 as t → ∞, respectively, where r 0 and R 0 are the responding basic reproduction numbers of avian influenza in the homogeneous bird world and mutant avian influenza in the homogeneous world, respectively.
Recently, much work with the basic reproduction number has been done to study the diseases dynamic properties. The basic reproduction numbers are usually defined by constants. But our definition of the basic reproduction numbers are time-dependent, and thus r F 0 (t) and R F 0 (t) can be utilized to predict spreading or vanishing of the avian influenza virus.
The sufficient conditions are given to determine the transmission of the avian influenza virus. Our results show that spreading or vanishing of the avian influenza is not only related to the basic reproduction numbers r F 0 (t) and R F 0 (t), but the initial infection. If r F 0 (0) < 1, R F 0 (0) < 1 and the initial infection value is small, the avian influenza disease vanishes, i.e., h ∞ − g ∞ < ∞, lim t→+∞ ( B i (·, t) C , H a (·, t) C , H m (·, t) C , H r (·, t) C ) = (0, 0, 0, 0) uniformly for x ∈ [g(t), h(t)] (see Theorem 5.9). It is shown that the mutant avian influenza spreads in the human world if r F 0 (0) < 1 and (see Theorem 6.1). If r F 0 (t 0 ) 1 for any t 0 0, then the avian influenza transmits in the bird world (see Corollary 7.2).
Nowadays, epidemic models have attracted much attention [14], especially, avian influenza model. The study of our work tries to describe the spreading process of the disease, and encourages people to take good strategies to prevent the avian influenza virus from transmitting to human world. There are some work to do, for example, the spreading speed when spreading happens.


extfieldsofstudy: Biology Medicine

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Characterization of VRC01, a potent and broadly neutralizing anti‐HIV mAb, produced in transiently and stably transformed tobacco

Summary The proposed clinical trial in Africa of VRC01, a potent broadly neutralizing antibody (bNAb) capable of neutralizing 91% of known HIV‐1 isolates, raises concerns about testing a treatment which will be too expensive to be accessible by the most important target population, the poor in under‐developed regions such as sub‐Saharan Africa. Here, we report the expression of VRC01 in plants as an economic alternative to conventional mammalian‐cell‐based production platforms. The heavy and light chain genes of VRC01 were cloned onto a single vector, pTRAk.2, which was transformed into Nicotiana benthamiana or Nicotiana tabacum using transient and stable expression production systems respectively. VRC01 has been successfully expressed transiently in plants with expression level of approximately 80 mg antibody/kg; stable transgenic lines expressing up to 100 mg antibody/kg were also obtained. Plant‐produced VRC01 from both systems showed a largely homogeneous N‐glycosylation profile with a single dominant glycoform. The binding kinetics to gp120 IIIB (approximately 1 nm), neutralization of HIV‐1 BaL or a panel of 10 VRC01‐sensitive HIV‐1 Env pseudoviruses of VRC01 produced in transient and stable plants were also consistent with VRC01 from HEK cells.

Introduction
Human immunodeficiency virus infection/acquired immunodeficiency syndrome (HIV/AIDS) is a global pandemic and one of the leading causes of deaths in the world (WHO, 2008). The worst affected region is sub-Saharan Africa, accounting for nearly 70 per cent of all people living with HIV. There is currently no cure or effective vaccine for HIV/AIDS. Current treatment protocols involve antiretroviral therapy (ART), a combination of three or more antiretroviral (ARV) drugs which control viraemia, thus increasing the survival rate of HIV-infected individuals (May and Ingle, 2011). ARV drugs can also be used to reduce the chance of HIV transmission through 'treatment as prevention' (TasP) (WHO, 2012a). Otherwise, ART can be used by noninfected individuals topically as vaginal or rectal microbicides (Shattock and Rosenberg, 2012) or as pre-or postexposure prophylaxis (PrEP or PEP) (WHO, 2012b;WHO and ILO, 2007).
Life-long treatment or prevention using ARV drugs is expensive, between US$10 000 and US$12 000 per year per person in the USA (Bartlett, 2006) as it involves sustained periods of drug delivery. Worldwide treatment coverage of people requiring access to ART, currently at 54 per cent (UNAIDS, 2012), still falls below the target goal of 80 per cent (UN, 2011;WHO, UNAIDS and UNICEF, 2007). Moreover, long-term use of ARV drugs can also result in toxicities with unpredictable effects on health as well as development of drug resistance and viral rebound if the treatment regimen is not adhered to (Barbaro, 2006;Barbaro et al., 2005;Chen et al., 2013;Rusconi et al., 2007). Therefore, the search continues for additional therapeutic modalities to complement or replace current ARV drugs (Arts and Hazuda, 2012).
Broadly neutralizing antibodies (bNAb) constitute a rare class of antibodies which are effective against a broad range of HIV strains Li et al., 2007Li et al., , 2009Simek et al., 2009). They naturally develop in 15%-25% of individuals after many years of infection. Most of the neutralizing antibodies, including bNAbs, disrupt viral replication by immune exclusion but there are also evidence to suggest viral neutralization by Fc-mediated antibody activities (Overbaugh and Morris, 2012). Since 2009, a new generation of bNAbs, which are more potent and broadly neutralizing compared with previous HIV-1 mAbs, has been identified (see Chen et al., 2013 for review). This new generation of bNAbs holds much promise for HIV-1 therapy and prevention. Recently, Klein et al. (2012) has shown that a combination of five new-generation bNAbs can effectively control HIV-1 infection and suppress viral load to levels below detection in a humanized mice model. The antibody cocktail has longer half-life compared with current ARV drugs and can control viraemia for an average of 60 days after therapy was discontinued. There are also plans for a clinical trial to test the new-generation bNAbs to prevent the spread of HIV-1 in high-risk adult males (Wadman, 2013). VRC01, one of these new generation broadly neutralizing IgG1, was obtained through the use of resurfaced antigenic probes to select for B cells (obtained from a HIV-1 infected donor) that express bNAbs directed at HIV-1 CD4 binding sites . It blocks viral entry by partially mimicking the interaction of the CD4 receptor with HIV-1 gp120 envelope glycoprotein . In vitro neutralization tests have shown that VRC01 is capable of neutralizing 91% of known HIV-1 isolates . The antibody, when applied as a topical gel, also protected against HIV-1 infection in vaginally challenged humanized mice (Veselinovic et al., 2012). Moreover, in the preclinical testing conducted in nonhuman primates, preliminary results showed that animals receiving VRC01 were protected against mucosal challenge with a chimeric simianhuman immunodeficiency virus (SHIV) compared with the control group (Pegu et al., 2011).
An investigational new drug application to the US Food and Drug Administration is currently underway for VRC01 (Lopez et al., 2013). The investigators proposed that VRC01 will be first safety-tested in adults and infants in the USA. It was then hoped that subsequent trials will be carried out in African countries such as Malawi, Tanzania and Uganda to investigate VRC01 as a prophylactic to complement ART in the prevention of mother-tochild transmission of HIV-1 via breastfeeding. The proposal for the clinical trial raised issues about testing a treatment which would not be affordable to its target population in low-income countries, as monoclonal antibody therapies are usually very expensive (Lopez et al., 2013;Wadman, 2013).
Plants offer an attractive alternative to conventional mammalian-cell-culture-based platforms for recombinant protein production, including monoclonal antibodies. In recent years, technology developments have resolved early difficulties with expression levels (Fischer et al., 2012). Furthermore, industry and regulatory concerns regarding cGMP compliant manufacture for human use have been addressed, with regulatory approvals for clinical trials in the UK (J. Ma, pers. comm.) and the USA (J. Butler, pers. comm.). Here, we compare transient and stable transgenic plant production platforms to manufacture new-generation bNAbs against HIV-1, using VRC01 as an example. VRC01 light and heavy chain genes were cloned into a single vector and transformed into tobacco plants. We report the successful production of VRC01 in tobacco using both methods. The plant-produced VRC01 antibodies possess a largely homogenous glycosylation pattern, and we have also investigated their binding kinetics. Furthermore, VRC01 from transiently and stably transformed plants neutralizes HIV-1 isolates across four different clades with neutralizing breadth and potency consistent with VRC01 from HEK cells. These developments now offer hope that the specific advantages of plant production platforms-namely low manufacturing entry cost, large scalability and simple technology amendable to transfer to developing countriesmay soon be realized.
For transient expression, Agrobacterium harbouring the T-DNA constructs were delivered into Nicotiana benthamiana via vacuum infiltration (Kapila et al., 1997). Twelve individual plants were used. The expression levels of 6-day postinfiltrated (dpi) N. benthamiana leaves (approx. 5-7 g) from each plant were determined using an ELISA designed to detect fully assembled IgG. The experiment was performed twice. The average expression level was 81 mg/kg fresh weight VRC01 mAb before purification.
pTRAk.2-VRC01 was also used to generate stably transformed plants through Agrobacterium co-cultivation of Nicotiana tabacum cv. Petit Havana SR-1 leaf discs (Horsch et al., 1985). Thirty-five primary transformants (T 0 ) were tested using ELISA set up to detect only full-length IgGs. The highest producing expressed at 100 mg/kg fresh weight (data not shown). This T 0 plant line was self-fertilized to generate T 1 plants. 30 T 1 plants were screened and the expression levels averaged about 130 mg/kg (unpurified; Table 1). The yield (before purification) of the VRC01 obtained from the stably transformed T 1 generation was not significantly different (P < 0.05) from the expression levels from N. benthamiana plants transiently transformed with pTRAk.2 (Table 1).

PAGE and Western blot analysis of mAb integrity
Coomassie-stained SDS-PAGE gels of VRC01 purified from transient and transgenic plants are shown in Figure 1a. In both cases, there is a major band at~150 kDa in the nonreduced samples that corresponds to the full-length IgG band identified in the positive control human IgG sample (top arrow). The identity of this band was confirmed by Western blot, as it was detected by both antiheavy and light chain antisera (Figure 1b, d). In all these antibody samples (transient, stable and positive IgG control), there were numerous smaller bands, which are likely to be degradation products as they were also immunoreactive in the Western blot. The stably expressed VRC01 appeared to demonstrate slightly more degradation, with prominent bands at 100 kDa and~50 kDa. Both of these bands were reactive with the antilight chain reagent in Western blot ( Figure 1d). Under reducing conditions, the bands resolved into a~50-kDa band and a~25-kDa band ( Figure 1a). Identical bands were observed for the HEK-cells-derived version of VRC01. These bands were identified as heavy and light chains by Western blotting (Figure 1c, e).

Glycopeptide analysis
The analysis of heavy and light chain sequences of VRC01 using EssembleGly (Caragea et al., 2007) revealed two potential N-linked glycosylation sites: one in the heavy chain C H 2 region at N 297 and one in the FR3 of the light chain V L region ( Figure 2a).
Glycan addition at both sites in VRC01 derived from HEK cells, transient plants and transgenic plants were confirmed using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) after S-carbamidomethylation and trypsin/GluC digest (Figure 2b-d). The deconvoluted spectra of the glycopeptide elution range were used for identification of N-glycan structures. HEK-cells-derived VRC01 was shown to have GnGnF 6 as the predominant glycan structure on the heavy chain, with lesser amounts of GnAF 6 , AAF 6 and GnGnF 6 bi structures ( Figure 2b, upper panel, nomenclature as used at www.proglycan.com). On the light chain, there was greater diversity of glycans with at least ten structures identified ( Figure 2b, lower panel). In contrast, the plant versions of VRC01 (which were similar), both have GnGnXF 3 as the major glycan structure, with MGnXF 3 and MMXF 3 as the two other major glycans (Figure 2c, d). This was observed in both the heavy and light chains. For the light chain, the MMXF 3 glycan was slightly more prevalent in stable expression of the antibody (Figure 2d, lower panel).

HIV-gp120 binding kinetics
To evaluate the antigen-binding kinetics of plant-produced VRC01, binding of HIV-1 envelope glycoprotein gp120 IIIB to VRC01 mAb captured on a protein A-coated CM5 sensor chip was measured by surface plasmon resonance (SPR). A preliminary direct ELISA confirmed that plant-derived VRC01 mAb binds to gp120 (data not shown). Protein A was first immobilized onto a CM5 sensor ship by amine coupling. Then, plant-or HEK-cellsderived VRC01 was captured on to the chip surface. Purified recombinant gp120 was injected over the chip at 20 lg/mL. The binding response of VRC01 from transient and stable plants as well as from HEK cells to gp120 molecules was fitted with a 1 : 1 Langmuir model after subtraction of the response from the blank flow cell and protein A-antibody only controls ( Figure 3a). The equilibrium dissociation constant (K D ) of the interaction between plant VRC01 and gp120 was calculated to be 1.68 AE 0.37 and 1.39 AE 0.62 nM, respectively, for transient and stable plantderived VRC01 compared with 0.66 AE 0.05 nM from VRC01 from HEK cells (Figure 3b). The association and dissociation constants of the SPR analyses are also shown in Figure 3b. There were no significant differences between the results (P < 0.05). Furthermore, there were no differences in relative antigen-binding activity (rela) between the two plant-derived VRC01 and HEK-cells-derived VRC01.

TZM-bl-neutralizing assays
We initially performed HIV neutralization assays comparing VRC01 produced in transient and stable plants and in HEK cells in inhibiting TZM-bl cell infection by HIV-1 BaL. The antibodies were first incubated with HIV-1 BaL at a range of concentrations before being added to TZM-bl cells containing  (Figure 4b). The IC 50 titres of VRC01 from transient plants were concordant with the HEK-cellsproduced VRC01 mAb. VRC01 from stable plants also neutralized all pseudoviruses, but appeared to be slightly less potent.

Discussion
Broadly neutralizing antibodies (bNAbs) have different properties compared with small molecule antiretroviral (ARV) drugs and represent an opportunity for alternative or complementary prophylactic and therapeutic approaches. For example, they are highly specific, interact and neutralize the pathogen directly and also possess a longer half-life in vivo (Klein et al., 2012). Anti-HIV bNAbs can be used in conjunction with small molecule ARV drugs in a multidrug combination, similar to antitumour (Scott et al., 2012) or antimicrobial (Pachl et al., 2006) combined therapy, to counter drug-resistant HIV-1 strains and viral escape (Pirrone et al., 2011). Furthermore, they can also be used for individuals intolerant to current ARV drugs. However, biologics are costly. The top 15 mAbs and Fc fusion products cost~$8 000 per gram in 2008 (Kelley, 2009). Prophylactic possibilities include vaginal or rectal microbicides, pre-exposure prophylaxis (PrEP), postexposure prophylaxis (PEP) and perinatal intervention. Furthermore, if mAbs are to be used in antiretroviral applications, several antibodies might be needed in combination as an antibody cocktail. Considering the long-term, sustained usage of ARV drugs in the treatment and prevention of HIV/AIDS (WHO, 2006), it is therefore imperative to look for less expensive and more scalable production platforms which can be used to replace or supplement current mammalian-cell-culture-based systems (Beck et al., 2010).
Plants were first proposed as a manufacturing platform for monoclonal antibodies over 20 years ago (Hiatt et al., 1989). Initial optimism for low-cost high-volume production was blunted both by technical limitations of the early technology as well as doubts over public acceptability of the new biotechnology. In addition, acceptance by the industry was affected by widespread concerns over the pharmaceutical regulatory compliance by plant-based manufacturing systems. The technical issues of recombinant protein expression levels have been addressed through a variety of means (Desai et al., 2010), including gene codon optimization (Batard et al., 2000;Rouwendal et al., 1997), expression vector design (Gleba et al., 2007;Sainsbury et al., 2009Sainsbury et al., , 2010, improved screening strategies of potential production lines, optimizing plant cultivation (Colgan et al., 2010) and plant breeding (Hood et al., 2002).
In 2012, the first plant-derived pharmaceutical product, Elelyso TM by Protalix BioTherapeutics, was approved by the FDA to treat Type I Gaucher's disease (Edney, 2013). Regulatory approvals have also been granted for clinical trials of, among others, recombinant human insulin produced in transgenic safflower, non-Hodgkins' lymphoma vaccine (comprising of monoclonal antibodies possessing the B-cell lymphoma idiotype) expressed transiently in N. benthamiana, and in 2011, the anti-HIV monoclonal antibody 2G12 produced in transgenic N. tabacum (Ma, J.K-C., Drossard, J., Lewis, D., Altmann, F., Boyle, J., Christou, P., Cole, T., Dale, P., van Dolleweerd, C.J., Isitt, V., Katinger, D., Lobedan, M., Mertens, H., Paul, M.J., Rademacher, T., Sack, M., Sparrow, P., Stiegler, G., Stoger, E., Twyman, R.M., Vcelar B. and Fischer R., pers. comm.). It is thus evident that recombinant pharmaceuticals manufacturing in plants are feasible. The focus now shifts back to target products and applications where plant manufacture is most desirable. Although lower cost of goods is a desirable feature of Nicotiana-based manufacturing, the potential for low cost of entry to clinical trials (a result of reduced early capital costs) may be an additional and significant driver. Of equal importance is the potential scalability of manufacture, which opens up prophylactic and/or therapeutic interventions that were previously unattainable.
HIV-neutralizing antibodies are therefore an obvious target for manufacture in plants. They would almost certainly be required at a very large scale, and cost of goods may become an important consideration for cocktail products containing two, three or more active drug products. Very recently, two groups reported production of VRC01 mAb in plant transient expression systems (Hamorsky et al., 2013;Rosenberg et al., 2013). Hamorsky et al. (2013) devised a novel tobamovirus replicon vector in N. benthamiana, while Rosenberg et al. (2013) reported transient expression using the same pTRAk vector used here in both N. benthamiana and N. tabacum. Both groups demonstrated successful expression of functional VRC01 of levels equivalent to those achieved here. In this study, we have demonstrated VRC01 production by stable expression in transgenic plants.
The finding that the expression levels in stable transgenics and transient expression were quite similar was unexpected, as previous literature has generally indicated much higher levels of expression using transient systems (Marillonnet et al., 2005;Sainsbury and Lomonossoff, 2008). Interestingly, with a second transient expression system CPMV-HT (Sainsbury and Lomonossoff, 2008), similar levels of expression were also obtained (data not shown). In the case of HIV bNAbs, the use of a highly efficient transgenic system may be extremely advantageous, because the simplified manufacturing process could be performed in resource-poor regions, especially those that have an urgent interest in addressing HIV transmission.
The transgenic plant lines generated here expressed VRC01 with no additional sequences (such as the KDEL tags used by Rosenberg et al.) or residual amino acids (as left by the kex2p-like cleavage strategy used by Hamorsky et al.). These transgenic plant lines have now entered a breeding programme to achieve homozygosity and establish genetic and phenotypic stability, which is the initial requirement for developing a master seed bank, leading eventually to GMP compliance (EMEA, 2008). VRC01 has two potential N-glycosylation sites in the heavy chain hinge region and Framework 2 of the light chain. Glycoanalysis of HEK-cells-derived VRC01 has not been published and this is the first report of VRC01 glycosylation in plants. We demonstrated that both potential N-glycosylation sites are utilized, in all the expression systems. Expression in plants generally resulted in a less heterogeneous mixture of glycoforms, as compared with HEK cells, with the typical GnGnXF 3 complex glycan dominant, particularly in transiently expressed VRC01. Mono-glycosylation of monoclonal antibodies has been demonstrated for one other antibody produced in plants, the anti-HIV mAb 2G12 (Forthal et al., 2010;Sainsbury et al., 2010), but not to our knowledge in other expression systems. In terms of product quality control, the ability of plants to produce highly homogeneous drug product could become an important manufacturing advantage. In cell-mediated anti-HIV activity, Forthal et al. (2010) reported that plant-produced 2G12 with the GnGnXF 3 glycoform performed as well as 2G12 produced in CHO-cells in terms of interaction of the antibody Fc region with FccRI, FccRIIa and FccRIIb receptors as well as antibody-dependent cell-mediated virus inhibition (ADCVI). 2G12 with plant-or mammalian-type core fucose did have reduced binding to FccRIIIa, and its potency in ADCVI assays could be enhanced by removal of the fucose residue. However, Rosenberg et al. (2013) showed that the removal of the same core a1,3-fucose from plant VRC01 did not increase its ADVCI potency. There is no evidence that plant glycans expressed on mammalian antibodies are immunogenic in humans. Plant-derived 2G12 with the GnGnFX 3 glycoform was administer vaginally to human volunteers with no adverse effects or immunogenicity (Ma, J.K-C., Drossard, J., Lewis, D., Altmann, F., Boyle, J., Christou, P., Cole, T., Dale, P., van Dolleweerd, C.J., Isitt, V., Katinger, D., Lobedan, M., Mertens, H., Paul, M.J., Rademacher, T., Sack, M., Sparrow, P., Stiegler, G., Stoger, E., Twyman, R.M., Vcelar B. and Fischer R., pers. comm.). That said, it is encouraging to note the advances that have been made in glycoengineering in plants (Castilho and Steinkellner, 2012;Castilho et al., 2013;Strasser et al., 2008Strasser et al., , 2009 and the potential for producing antibodies with 'mammalian' glycans. Interestingly, when mAb 2G12 was expressed with altered glycosylation, there was no significant increase in glycoform heterogeneity (Forthal et al., 2010;Strasser et al., 2008).
The affinity (K D ) of VRC01 produced in transient and stable plants to the HIV-1 envelope glycoprotein gp120 IIIB was 1.68 and 1.39 nM, respectively, as determined by SPR analysis. There was no significant difference (P < 0.05) in the binding affinities between plant-and HEK-cells-derived VRC01. Rosenberg et al. (2013) and Hamorsky et al. (2013) have also compared the binding of plant-and mammalian-cell-produced VRC01 to gp120 using capture assays with immobilized mouse anti-human IgG or protein A. Neither group found any difference in the binding affinities of plant-and mammalian-cell-produced VRC01. Zhou et al. (2010) obtained a K D of 5.76 nM when they investigated the binding of HEK-cells-produced VRC01 to 93TH057 gp120 using a direct assay. Furthermore, the gp120 binding activities of both plant-derived VRC01 were very similar to VRC01 from HEK cells. This is in contrast to Hamorsky et al. (2013) who reported 75%-83% relative activity of transiently expressed VRC01 when compared to HEK-cells-derived VRC01.
Neutralization by plant-derived VRC01 was compared with HEK-cells-derived VRC01 across 11 HIV-1 virus strains, including representatives from clades A, B, C and G. The results were concordant between VRC01 from transient plant and HEK cells, demonstrating functional equivalence for the plant-derived antibody. The IC 50 measured in the pseudotype assays suggest a reduction in potency for VRC01 from stable transformants. This is not likely to reflect qualitative differences between the VRC01 from various sources. The SDS-PAGE and glycoanalysis suggested increased proteolytic degradation of the stably produced VRC01 sample. Indeed, this sample had been subjected to at least three freeze-thaw cycles before pseudotype neutralization analysis (compared with 1 for the transiently expressed VRC01). No difference in neutralization of HIV-1 BaL was observed, using material that had undergone two less freeze-thaw cycle. Thus, the slight increase in IC 50 observed is likely to be due to error in calculation of the true concentration of full-length, functional IgG in the tested sample. Degradation of antibody might be influenced by the expression platform, but no differences were observed previously when comparing mAb expression by transgenic or transient expression (V. Hehle, PhD thesis, University of London). Edman degradation experiments using three other human monoclonal antibodies from stably transformed plants have suggested that the~100-kDa bands are degradation products (Hehle et al., 2011;V. Hehle, PhD thesis) likely to represent (Fab') 2 -like fragments. The~50-kDa band could be light chain dimers. In fact, light chain excess appears to be required for maximal expression of intact full-length IgG in plants (Nuttall et al., 2002). The components of these degradation products may account for the excess of the 25-kDa band compared with the 50-kDa band on the reduced PAGE gel (Figure 1a, middle panel). The results have indicated that further work will be needed to finalize a downstream purification process and to characterize the stability in storage of highly purified plantderived VRC01. They also highlight the importance of avoiding repeated freeze-thaw cycles.
In summary, the new-generation bNAb VRC01 was successfully produced using both stable and transient production platforms. This was achieved using a single vector that contains both the light and heavy chain genes under separate promoters. Intact plant-produced VRC01 was expressed at approximately 80 mg/kg fresh weight using transient expression platforms, and stable lines expressing in excess of 100 mg/kg VRC01 were also obtained. In both cases, the expression levels are significantly higher than that reported for the 2G12 monoclonal antibody that was advanced to phase I clinical trial (www.pharma-planta.net). Further work would be needed to develop appropriate downstream processing/purification protocols, and the efficiency of these protocols will determine the final productivity of the expression system. Plant-produced VRC01 showed N-glycosylation profiles that were similar to HEK-cells-derived VRC01, but more homogeneous. GnGnXF 3 was the single dominant glycan structure. The binding kinetics of VRC01 produced in stable and transient plants are consistent with values in the published literature for VRC01 from HEK cells. Furthermore, the neutralizing breadth and potency of plant-produced VRC01 matched that of HEK-cells-produced VRC01 across a panel of HIV-1 virus and pseudoviruses from four different clades. This was also a first demonstration of VRC01 production in stably transformed, transgenic lines, a manufacturing platform which is readily transferable to resource-poor regions, and for which a regulatory pathway for cGMP compliant production has already been established in Europe (EMEA, 2008).

Experimental procedures
Cloning of VRC01 heavy and light chain genes The heavy (gamma) and light (kappa) chain genes of the broadly neutralizing antibody VRC01 were plant-codon-optimized using GeneArt proprietary software (Geneart, Regensburg, Germany) and provided by MAPP Biopharmaceuticals (San Diego, CA).
For cloning into the binary expression vector pTRAk.2 (Sack et al., 2007;van Dolleweerd et al., in preparation), the genes were amplified using the common forward primer 5′ GCGCCCATGGCTAACAAGCACCTGTCT-3′ and the reverse primer 5′GCGCTCTAGACTAGCACTCTCCCCTATTAAAAG-3′ (for light chain) or 5′GCGCTCTAGACTACTTACCAGGAGACAGA GAC-3′ (for heavy chain). Restriction sites used for cloning the gene in to pTRAk.2 are underlined and the start/stop codons are in bold. The amplified PCR products were cloned into BsmBI site (for heavy chain) or BsaI site (for light chain) of a single pTRAk.2 vector using NcoI and XbaI.

Stable transformation of Nicotiana tabacum
Stable transformation of N. tabacum leaf discs was performed as described by Horsch et al. (1985). Briefly, Agrobacterium tumefaciens strain GV3101 : pMP90(RK) transformed with pTRAk.2 harbouring VRC01 light and heavy chain genes was grown as above. After removal of medium by centrifugation, the pellet was resuspended in liquid MS (Murashige and Skoog, 1962) medium (Sigma, Gillingham, UK). Leaf discs (1 cm 2 ) of 6week-old N. tabacum cv Petit Havana SR-1 grown under sterile conditions were immersed in the bacterial suspension for 20 min. The leaf discs were then incubated for 2 days on shoot-inducing medium (SIM) consisting of MS medium, 0.1 lg/mL a-naphthaleneacetic acid (NAA; Sigma) and 1 lg/mL benzylaminopurine (BAP; Sigma) at 23°C. The leaf discs were then transferred to SIM with 500 lg/mL carbenicillin (Apollo Scientific, Stockport, UK) to eliminate the Agrobacterium and 100 lg/mL kanamycin to select for transformed cells. Regenerated shoots were rooted on MS medium with carbenicillin and kanamycin. Rooted plantlets were transferred to soil. Yields of rooted primary transformants (T 0 ) were determined by ELISA (see below) of plant crude extracts obtained from two leaf discs. Three of the highest yielding T 0 plants were allowed to self-pollinate to obtain the T 1 generation.
The highest yielding T 0 plant was also repropagated by tissue culture to obtain purified VRC01 antibody. Briefly, leaves of the plant were sterilized for 15 min in 15% sodium hypochlorite. 1-cm 2 leaf discs were cultured in SIM with 500 lg/mL carbenicillin and 100 lg/mL kanamycin. Regenerated shoots were rooted on MS medium before being transferred to soil. Leaves of the regenerated plants were harvested for protein purification after 2.5-3 months just before flowering.

Transient transformation of Nicotiana benthamiana
Vacuum infiltration of N. benthamiana leaves was performed as described by Kapila et al. (1997). Agrobacterium tumefaciens strain GV3101 pMP90RK transformed with pTRAk.2 harbouring VRC01 light and heavy chain genes was grown overnight in Luria-Bertani (LB) broth, 0.02 mM acetosyringone (Santa Cruz Biotechnology, Dallas, TX), 0.01 mM MES (Sigma), 100 lg/mL rifampicin (Apollo Scientific) and 50 lg/mL kanamycin (Apollo Scientific) at 28°C. After removal of the medium by centrifugation, the pellet was resuspended in infection solution containing 0.1 mM acetosyringone, 0.01 mM MES (Sigma) and 0.01 mM MgCl 2 (VWR International, Lutterworth, UK). The final OD 600 of the bacterial suspensions was adjusted to 0.3. Fully expanded leaves of 3.5week-old N. benthamiana plants were immersed in the bacterial suspension in desiccator attached to a vacuum system consisting of a vacuum pump (Vacuubrand GMBH, Brackley, UK). Vacuum was applied for 1 min at 100 mbar. The plants were then further grown in a controlled environment room at 25°C with 16/8 h light/dark cycle.

Immunoabsorbent assays (ELISA)
Five microgram per millilitre sheep anti-human IgG gamma heavy chain Fc region (The Binding Site, Birmingham, UK) or 1 lg/mL gp120 IIIB (NIH ARRRP) was coated for 2 h at 37°C. The plates were washed with three washes of wash buffer (water with 0.01% Tween-20). The plates were blocked with PBS with 5% skimmed milk powder at 37°C for 1 h. After three washes with wash buffer, the antibody samples (as crude plant extracts or pure protein) were incubated at 37°C for 2 h (all dilutions were done in blocking solution). The plates were washed three times with wash buffer. A 1 : 1000 dilution of peroxidase-conjugate goat anti-human kappa light chain (Sigma) was added for 1 h. Bound antibodies were detected using TMB substrate solution (3,3′,5,5′-tetramethylbenzidine (Sigma) in 25 mM citrate-50 mM phosphate buffer, pH 5.0). The colour reaction was stopped by addition of 2 M sulphuric acid, and the absorbance was determined at 450 nm using a Sunrise plate reader (Tecan, Theale, UK). Titrations of known human IgG1 kappa (Sigma) were used as standards to determine antibody concentration.

Protein purification
Vacuum infiltrated leaves were harvested at 6 days postinfection (dpi) for transient plants or just before flowering for stable plants.
The leaves were extracted with three volumes of extraction buffer (100 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA and 1 lM PMSF). The clarified crude extracts were purified using a mixed protein G-Sepharose/protein A-agarose (Sigma) column. The column was washed with 10 column volumes (CV) of binding buffer (20 mM sodium phosphate, pH 7.0) and the protein eluted with elution buffer (0.1 M glycine-HCl, pH 2.7). The eluted fraction was neutralized with 1 M Tris-HCl, pH 9.0 and then by buffer-exchange diafiltration using Amicon Ultra-15 (Molecular cut-off 100 kDa; Milipore, Ireland) or by dialysis using Slide-A-Lyzer Cassettes (Molecular cut-off 3.5 kDa; Thermo Scientific, USA) depending on the volume. Purified antibody concentration was determined by direct ELISA using titrations of known human IgG kappa (Sigma) as standards, reconfirmed with resolving titrations of purified antibodies against known concentrations of human IgG kappa on a NuPage 4%-12% Bis-Tris Coomassie gel (Life Technologies) and either with A280 measurements using NanoDrop 2000 (Thermo Scientific, USA) or with BCA protein assay (Pierce, USA) using known concentration of bovine serum albumin (BSA) as standards.

Glycopeptide analysis using LC-ESI-MS
Purified antibodies were reduced using 5 mM DTT in a 0.1 M ammonium bicarbonate buffer at 56°C for 45 min followed by S-carbamidomethylation using 25 mM iodoacetamide in a 0.1 M ammonium bicarbonate buffer at 25°C for 30 min. After precipitation using 80% acetone at À20°C for 45 min, the protein was digested with 0.2 lg trypsin in 0.1 M ammonium bicarbonate buffer overnight at 37°C. After deactivation of trypsin at 96°C for 6 min, the protein was digested with 0.2 lg endoproteinase GluC in 0.1 M ammonium bicarbonate buffer overnight at 37°C. Glycopeptide analysis was performed using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) as described previously (Pabst et al., 2012) with a few modifications: the gradient was developed from 1% to 80% acetonitrile over 60 min at a flow rate of 6 lL/min, using 0.3 formic acid buffered to pH 3.0 with ammonia as the aqueous solvent. Peptide identification was performed with maXis 4G ETD (Bruker, Germany) in positive ion mode (ion cooler transfer time: 100 ls, ion cooler prepulse storage time: 10 ls; low mass: 300.00 m/z, spectra rate: 1 Hz).

Surface plasmon resonance of antibody binding
The binding kinetics of plant-and HEK-cells-produced VRC01 to HIV-1 gp120 IIIB were determined using a Protein A capture assay (Floss et al., 2009). The assay was performed on a BIAcore X-100 instrument (GE healthcare, Chalfont St Giles, UK) at 25°C with buffer HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.05% surfactant P-20). Protein A (Sigma) was immobilized onto a CM5 chip to 3988 response units (RUs) with standard amine coupling. Purified VRC01 from plant or HEK cells was diluted in HBS-EP+ buffer to yield a similar capture level of 100 RUs. gp120 IIIB was injected at a concentration of 20 lg/mL for 3 min at the flow rate of 30 lL/min and allowed to dissociate for another 7.5 min before regeneration with 10 mM HCl for 1 min at the flow rate of 10 lL/min. Referenced and blanked sensorgrams were fitted with BIAcore Evaluation software using a 1 : 1 Langmuir model of binding. Each assay was performed in triplicate. The relative activity (rela) of the plantderived VRC01 was calculated in relation to the HEK-cells-derived VRC01 using the equation described by Floss et al. (2009) with slight modifications: the gp120 binding response (R t ) was taken at 190 s after the start of injection.

HIV-1 neutralization assays
HIV-1 BaL neutralization assay was performed as described by Wei et al. (2003) and Montefiori (2005 BaL was incubated with dilutions of purified plant-or HEK-cellsproduced VRC01 for 1 h at 37°C in a total volume of 100 lL. Plant-and HEK-cells-produced VRC01 were titrated twofold seven times in triplicate starting at 10 lg/mL. All dilutions were performed in growth medium [DMEM with 10% foetal calf serum (FCS), penicillin-streptomycin (100 units/mL and 100 lg/mL, respectively) and 2 mM L-glutamine]. The mixture were then added to 100 lL TZM-bl cells expressing CD4, CCR5 and firefly luciferase gene under the control of HIV long-terminal repeat sequence grown in growth medium preseeded at a concentration of 5 9 10 5 cells/mL. Cells plus virus positive control and a cells only background control were also added. After a 24-h incubation in 37°C, 5% CO 2 followed by addition and incubation with lysis buffer, the cell lysate was removed from each well and mixed in a 1 : 1 ratio with Bright Glo reagent (Promega, Southampton, UK) in a 96-well white solid plates for luminescence measurements. The percentage reduction in relative light units (RLU) was calculated relative to the RLU of the 'virus plus cells' positive control. The resulting curve was plotted and analysed in GraphPad Prism.

Statistical methods
All statistical analyses were performed using GraphPad Prism 6 software (GraphPad, San Diego) by one-way analysis of variance (ANOVA) with the significance level set at P < 0.05. This is followed by Tukey-Kramer post hoc analysis.


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Detection of Quantitative Trait Loci (QTL) Related to Grilsing and Late Sexual Maturation in Atlantic Salmon (Salmo salar)

In Atlantic salmon aquaculture, early sexual maturation represents a major problem for producers. This is especially true for grilse, which mature after one sea winter before reaching a desirable harvest weight, rather than after two sea winters. Salmon maturing as grilse have a much lower market value than later maturing individuals. For this reason, most companies desire fish that grow fast and mature late. Marker-assisted selection has the potential to improve the efficiency of selection against early maturation and for late sexual maturation; however, studies identifying age of sexual maturation-related genetic markers are lacking for Atlantic salmon. Therefore, we used a 6.5K single-nucleotide polymorphism (SNP) array to genotype five families from the Mainstream Canada broodstock program and search for SNPs associated with early (grilsing) or late sexual maturation. There were 529 SNP loci that were variable across all five families, and this was the set that was used for quantitative trait loci (QTL) analysis. GridQTL identified two chromosomes, Ssa10 and Ssa21, containing QTL related to grilsing. In contrast, only one QTL, on Ssa18, was found linked to late maturation in Atlantic salmon. Our previous work on these five families did not identify genome-wide significant growth-related QTL on Ssa10, Ssa21, or Ssa18. Therefore, taken together, these results suggest that both grilsing and late sexual maturation are controlled independently of one another and also from growth-related traits. The identification of genomic regions associated with grilsing or late sexual maturation provide an opportunity to incorporate this information into selective breeding programs that will enhance Atlantic salmon farming.

Introduction
The Atlantic salmon (Salmo salar) exhibits a great deal of variability in age and size at sexual maturation. This variation is observed both between and within strains and year classes (Garcia de Leaniz et al. 2007;Taranger et al. 2010). The timing of sexual maturation is controlled by a complex process that involves genetic and environmental components (Thorpe and Metcalfe 1998). Internal factors, such as age and lipid reserves, together with external factors, like light abundance, temperature, and food intake, seem to have an impact on the initiation of sexual maturation (Gardner 1976;Herbinger and Friars 1992;Simpson 1992). The interactions of these factors result in the enormous variability in the age of sexual maturation, especially in male Atlantic salmon, which are able to reach maturity at 1 to 7 years of age (Simpson 1992). There is also a genetic component to age at sexual maturation in Atlantic salmon, and estimates of heritabilities (h 2 ) for this trait in Atlantic salmon range widely from 0.09-0.17 Wild et al. 1994;Gjedrem 2000) to 0.39 (Gjerde and Gjedrem 1984) to 0.48 (Gjerde 1984). These observations strongly indicate that age at sexual maturity in Atlantic salmon is a heritable trait. Thus, it should be possible to select for Atlantic salmon that do not become sexually mature as grilse and also for late sexually maturing fish.
In salmon aquaculture, early sexual maturation can be a major economic problem. This is particularly costly in terms of feed and cage space if fish mature as grilse (i.e., after 1 year in a sea cage; Gjedrem 2000). Salmon maturing as grilse have a much lower market value than later maturing individuals (Saunders et al. 1983). The maturation process is energetically expensive, and this is reflected in a decrease in growth rate, lower meat quality, and increased mortality through susceptibility to pathogens (Gjerde 1984;Thorpe 1994). Late-maturing salmon are desired in commercial operations, and to reduce the frequency of grilsing, early maturing Atlantic salmon are discarded as potential breeding candidates (Gjedrem 2012). Selection for the economically important production traits of fast growth and late sexual maturation has been considered problematic as it has been suggested that there is a correlation between the phenotypes of fast growth and early sexual maturation (Thorpe et al. 1983).
Marker-assisted selection has the potential to improve the efficiency of selection for traits such as age of sexual maturation and growth in Atlantic salmon breeding. Genomic regions linked to sexual maturation have been identified in other salmonid species such as rainbow trout and Arctic charr (Easton et al. 2011;Haidle et al. 2008;Küttner et al. 2011;Martyniuk et al. 2003;Moghadam et al. 2007). However, such studies in Atlantic salmon are lacking. The identification of genetic markers independently related to either early or late sexual maturation, especially in male Atlantic salmon, would enable the implementation of selective breeding based on improved genetic selection practices by identifying animals with favorable genotypes. We previously identified QTL for growth at different stages of the production cycle in five families from the Mainstream Canada broodstock (Gutierrez et al. 2012). Here we report QTL for grilsing and late sexual maturation in the same families and show that the genomic locations of these QTL are independent of one another as well as the QTL associated with growth.

Mapping Families and Phenotype Data
Families were part of a commercial broodstock program developed by Mainstream Canada and based on the Mowi strain of Atlantic salmon, which was derived from a breeding program established using Norwegian populations (Gjedrem et al. 1991). In November/December 2005, 130 single-pair mating families were established. At the fry stage (February 2006), 120 offspring from each family were pooled (15,600 fish in total) and grown communally at the Oceans Farms Hatchery, Vancouver Island. In September/October 2006, 5,000 of the fish were passive integrated transponder (PIT)tagged, and phenotypic measurements were taken until early 2009. Maturation times in Atlantic salmon were classified as: precocious (≤12 months of age), grilse (36 months of age, at first sea winter (SW)), normally maturing (48 to 60 months of age at second SW or third SW), and late-maturing fish (>60 months) (Gjedrem 2000;Taranger et al. 2010). Sex was recorded during the confirmation of maturation status. The sex of an individual in the late-maturing group could not be ascertained phenotypically; therefore, we predicted the sex of the late-maturing Atlantic salmon using a polymerase chain reaction test, developed by Eisbrenner et al. (2013) that amplifies two exons of the sdY gene, which has been shown to be the sex-determining gene in rainbow trout and probably other members of the Salmoninae (Yano et al. 2012(Yano et al. , 2013. It should be noted that the precocious Atlantic salmon that matured before 1 year of age were discarded during a standard grading procedure, and so no samples were available from this group.

DNA Extraction and Parental Assignments
These procedures were as described by Gutierrez et al. (2012). Briefly, DNA was extracted from the adipose fins of the PITtagged progeny and the parents used to produce the 130 families (Withler et al. 2005). The DNA was then genotyped using eight microsatellite markers, and parental assignment was carried out as described by Withler et al. (2007).

SNP Array and Linkage Mapping
The single-nucleotide polymorphism (SNP) data used for this analysis have been described previously (Gutierrez et al. 2012). Five families were selected for SNP genotyping at CIGENE, Norwegian University of Life Sciences, Ås, Norway, using an Atlantic salmon 6.5K Illumina iSelect SNP-array (Kent et al. 2009). Analyses were based on an Atlantic salmon linkage map, which contains ∼5,650 SNP markers and was constructed using genotyping data from 143 families comprising 3,297 fish ). This map contains 29 linkage groups, which were assigned to their specific chromosome number according to the nomenclature established by Phillips et al. (2009). All non-informative markers were removed from the datasets, and subsequently, independent male and female linkage maps were manually created for each family and adjusted to the distances on the SNP map of Lien et al. (2011). Given the differences between the sets of informative markers present in each family, for this study, we restricted the analysis to the 529 markers which are informative in all of the five families. These markers are distributed across all 29 Atlantic salmon chromosomes. We have previously commented on some of the possible reasons why only ∼10% of the markers were variable in all of the five families (Gutierrez et al. 2012). It may reflect the starting population for the Mainstream Canada broodstock or past selection (e.g., primarily for growth in terms of days in salt water to reach 5 kg), or it may reflect the nature of biallelic SNPs. We note that many QTL studies in salmonids have traditionally used microsatellite markers, and these studies tend to have employed fewer variable markers and also fewer families. Even when SNPs were used as the genetic marker, the overall number tested for QTL analysis has been less than 350, and not all of these were variable in each family. Therefore, we consider this study to be robust in terms of what is currently available in the salmonid aquaculture literature.

QTL Analyses
QTL analyses were performed using a regression-interval mapping method available through the GridQTL portlet (Seaton et al. 2006). GridQTL is a portlet environment (available at http:// www.gridqtl.org.uk/) that allows the analysis of extensive datasets. The datasets were analyzed as sib-pairs using the combined genotypic data from the 529 informative markers shared by all families. For the grilsing analysis, given the absence of female grilse, only the genotypic information from males was used. The phenotypic data were scored as a binary trait, i.e., male fish recorded as grilse were scored 0, and male fish recorded as maturing at second or third SW were scored as 1. The same procedure was used for the analysis of late sexual maturation, but both male and female fish were used in this case. Atlantic salmon that did not show evidence of gonadal development at 60 months were considered "late-maturing" and scored as 0. The results were illustrated using the female map as it provides a greater resolution than the male genetic map.
The percentage of phenotypic variance (PEV) explained by the QTL in the sib-pair analysis was calculated as a proportion of the phenotypic variance obtained from the observations. Fstatistic values were calculated at 1 cM intervals on each chromosome for each analytical approach to yield the most likely position of the QTL. Empirical chromosome-wide significance thresholds were determined by permutation tests (Churchill and Doerge 1994). The 10,000 permutations were performed at the chromosome-wide level in order to establish the F-value thresholds for a p<0.05 and p<0.01. F-critical values corresponding to 0.05>p>0.01 were considered as "suggestive" QTL, whereas those corresponding to p<0.01 were considered "significant" QTL. Those chromosomes which contained QTL that were found to be "significant" were tested for their significance at a genome-wide level of p<0.05 by performing a 1,000 permutation test.

QTL Analysis for Male Grilsing
As described previously (Gutierrez et al. 2012), five families were chosen from the 2005 year class of the Mainstream Canada selective breeding program for analysis. Each family contained from five to eight male grilse. No female grilse were observed in any of these families ( Table 1). As shown in Fig. 1 and Table 2, QTL analyses restricted to male grilsing revealed the presence of two statistically meaningful QTL, one on chromosome 10 (Ssa10) and the other on chromosome 21 (Ssa21). The QTL on Ssa21 was considered significant (p<0.01), whereas the other on Ssa10 only reached the suggestive level of significance (0.05>p>0.01). Neither of these QTL reached genome-wide significance, which is in keeping with the relatively small sample size as well as the rather low h 2 estimates in Atlantic salmon for age at sexual maturation (Gjerde 1984;Gjerde et al. 1994;Wild et al. 1994). The PEV explained by these QTL was 16.7% and 25% in Ssa10 and Ssa21, respectively.

QTL Analysis of Late Sexual Maturation
From the 195 individuals used in the analysis of late sexual maturation, 40 were fish that did not show gonadal development after their third SW. The sex of the individuals in this latter group was determined based on the presence of two of the four exons of the sdY gene (Eisbrenner et al. 2013). Seventeen of the late-maturing 40 fish were male and 23 were female (Table 1). The GridQTL analysis revealed the presence of only one statistically significant (p<0.01) QTL on chromosome 18 (Ssa18), which did not reach genome-wide significance, even though it was the only QTL found for this particular trait (Fig. 2). This QTL explained 14.7 % of the PEV associated with late sexual maturation in these Atlantic salmon families.

Discussion
Previous studies performed in rainbow trout and Arctic charr have shown an apparent link between QTL for sexual maturation and growth (Haidle et al. 2008;Martyniuk et al. 2003;Moghadam et al. 2007); however, our results indicate a different scenario for Atlantic salmon in the Mainstream Canada breeding program. Here we only detected two QTL associated with male grilsing in Atlantic salmon, on Ssa10 and Ssa21. Previously using the same families, Gutierrez et al. (2012) found genome-wide significant growth-related QTL on chromosomes Ssa02, Ssa07, Ssa13, Ssa09, Ssa17, and Ssa26. Although Gutierrez et al. (2012) found some suggestive evidence for growth-related QTL on Ssa10 and Ssa21, neither reached genome-wide significance, and they mapped to different regions of the chromosomes compared with the grilsing QTL. Moreover, the results of QTL analyses for growth and sexual maturation in the Mainstream Canada broodstock families indicate that sexual maturation is controlled by fewer genomic regions than growth in Atlantic salmon. Therefore, it should be possible to tease apart these traits genetically and so select for each independently using specific genetic markers. A different situation was observed for the QTL for late sexual maturation detected on Ssa18. To date, no growthrelated QTL have been reported on this chromosome in Atlantic salmon or on homologous chromosomes in other salmonids (Gutierrez et al. 2012).
Correlations between the phenotypes of growth (as estimated by body weight) and early sexual maturation have been reported in Atlantic salmon (Gjerde 1984;Gjerde et al. 1994;Wild et al. 1994). Moreover, considerable genetic variation in age at sexual maturity has been described in farmed fish species. For example, in Atlantic salmon h 2 estimates range from 0.09 to 0.17 (Gjerde 1984;Gjerde et al. 1994;Wild et al. 1994) to 0.39 (Gjerde and Gjedrem 1984) to 0.48 (Gjerde 1984) whereas in rainbow trout h 2 estimates range from 0.12 to 0.35 (Gjerde and Schaeffer 1989;Kause et al. 2003). Nevertheless, selective breeding programs have been effective in increasing body size while also controlling undesired early sexual maturity in farmed fish (Gjedrem 2000), giving clues that these traits are influenced by different genes. Our previous results showed that growth-related QTL were spread across the Atlantic salmon genome (Gutierrez et al. 2012); however, none of the QTL associated with grilsing or late sexual maturation were found in the same chromosomal positions and with the same statistical significance indicating that different genes are associated with these traits.
Comparative genetic mapping in salmonid species has revealed homologous regions of the genomes of Atlantic salmon, rainbow trout, and Arctic charr (Danzmann et al.   (Phillips et al. 2009) and Arctic charr linkage group 18 (AC-18) (Danzmann et al. 2005), whereas Ssa10 corresponds to RT-8q and RT-27q (equivalent to chromosomes 5q and 2q, respectively) (Phillips et al. 2009) and Arctic charr linkage groups 4, 16, and 40 (AC-4, AC-16, and AC-40) (Danzmann et al. 2005). Genome-wide significant QTL for early male sexual maturation were mapped to RT-8, RT-17, and RT-24, whereas chromosome-wide QTL for this trait was found on RT-3 and RT-19 (Haidle et al. 2008). Martyniuk et al. (2003) did report an association (p=0.043) between microsatellite locus OmyRGT1TUF on RT-5 and precocious maturation and noted that one allele was associated with both a higher body mass and precociousness. However, this association was not considered significant after a sequential Bonferroni correction. Similarly, no p values less than 0.01 were identified for microsatellite marker alleles segregating with male sexual maturation in Arctic charr, although SSOSL32 on AC-4 gave a suggestive value of 0.014 (Moghadam et al. 2007). It is believed that sexual maturation in salmonids depends on the ability of the fish to reach certain developmental thresholds during a "critical period" over the winter/spring (Adams and Thorpe 1989;Thorpe 1994), in a direct relationship with somatic growth and/or energy storage during this period (Taranger et al. 1999). Further investigation of genotype-environment interactions are required to improve Atlantic salmon breeding programs. Photoperiod manipulation using artificial light exposure has proven effective as a means of preventing Atlantic salmon from reaching an anticipated sexual maturation (Endal et al. 2000;Oppedal et al. 2006), and this procedure is used with many other farmed fish (reviewed by Taranger et al. 2010). A clear link between feeding and age of sexual maturation has been described in salmonids (Shearer et al. 2006;Silverstein et al. 1998;Taranger et al. 2010;Thorpe et al. 1990), suggesting that by restricting rations, energy storage and, in particular, lipid stores decrease, thereby delaying sexual maturation (Duston and Saunders 1999;Rowe et al. 1991;Shearer et al. 2006;Shearer and Swanson 2000). This also explains the increasing use of tetradecylthioacetic acid to reduce early sexual maturation in Atlantic salmon (Arge et al. 2012) as it affects the β-oxidation of fatty acids, reducing levels of plasma lipids and adipose lipid stores, and enhancing transport of fatty acids to the liver.

Candidate Genes
To identify candidate genes associated with the traits of early or late sexual maturation, we made use of the currently available information from the Atlantic salmon genome sequencing project (Davidson et al. 2010), which is publicly available at ASalBase (www.asalbase.org). Most of the SNPs in the 6. 5K array correspond to an expressed sequence tag (EST) or are linked to a specific genomic sequence, and these sequences were assigned to a specific whole genome shotgun (WGS) contig by sequence similarity searches. WGS contigs were then annotated using an in-house annotation pipeline (trutta.mbb.sfu.ca) (see Table 3).
There are four SNPs linked to the QTL associated with grilsing on Ssa10, of which two could be linked to two genes by a BLASTn search (Altschul et al. 1990) and subsequent sequence annotation. These genes were a leukocyte elastase inhibitor (SERPIN-like) and a GDP-L-fucose synthase-like protein. We cannot speculate how these genes may be related to grilsing in Atlantic salmon. In the case of the SNPs linked to the grilsing QTL on Ssa21, one is associated with a gene encoding a formimidoyl transferase-cyclodeaminase, a protein related to metabolic processes. Two other SNPs (from the same EST) identified E3 ubiquitin-protein ligase (UBR3), a protein that has been shown to have a regulatory role in sensory pathways, including olfaction in mammals (Tasaki et al. 2007). The QTL detected for the late-maturation trait on Ssa18 was linked to two SNPs, but only one of them could be related to a known gene, namely, myopalladin-like gene. Studies in pig and cattle suggest  that this gene is associated with meat quality and carcass traits including fat content (Chong et al. 2007;Jiao et al. 2010).

Conclusions
In this study, we were able to identify a limited number of QTL associated with age at sexual maturation in Atlantic salmon. Moreover, our results suggest that these traits are controlled by genes independently from growth-related traits. Age at sexual maturation is a trait that has been heavily selected and improved after several generations of selective breeding based on family selection and individual selection (Gjedrem 2000;Gjerde 1984;Gjøen and Bentsen 1997). However, these improvements have been based on phenotypic observations, and thus, the identification of genetic markers and genomic regions associated with grilsing and late sexual maturation provides new resources for Atlantic salmon selective breeding programs.