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1 Adenoviral expression of a bispecific VHH-based neutralizing agent targeting protective 2 antigen provides prophylactic protection from anthrax in mice 3 4 5 6 7 8 Mahtab Moayeri1, Jacqueline M. Tremblay2, Michelle Debatis2, Igor P. Dmietriev3, Elena A. 9 Kashentseva3, Anthony J. Yeh4, Gordon Y.C. Cheung4, David T. Curiel3, Stephen Leppla1, Charles B. 10 Shoemaker2# 11 12 13 14 15 1Microbial Pathogenesis Section, Laboratory of Parasitic Diseases, National Institute of Allergy 16 and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA. 17 2Department of Infectious Disease and Global Health, Tufts Cummings School of Veterinary 18 Medicine, North Grafton, MA, USA. 19 3Department of Radiation Oncology, Washington University, St. Louis, MO, USA. 20 4Pathogen Molecular Genetics Section, Laboratory of Bacteriology, National Institute of Allergy 21 and Infectious Disease, National Institutes of Health, Bethesda, MD, USA. 22 23 24 #Corresponding author 25 Corresponding author: Dr. Charles B. Shoemaker 26 Department of Infectious Disease and Global Health, Tufts Cummings School of Veterinary 27 Medicine, 200 Westboro Rd, North Grafton, MA 01536 28 E-mail address: charles.shoemaker@tufts.edu 29 Tel. 508-887-4324; Fax 508-839-7911 30 31 32 33

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Running title: Adenovirus VNA gene therapy against anthrax 36

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CVI Accepted Manuscript Posted Online 6 January 2016Clin. Vaccine Immunol. doi:10.1128/CVI.00611-15Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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Abstract (Limit 250) 38

Bacillus anthracis, the causative agent of anthrax, secretes three polypeptides which form the 39

bipartite lethal and edema toxins (LT, ET). The common component in these toxins, protective 40

antigen (PA), is responsible for binding to cellular receptors and translocating the lethal factor 41

(LF) and edema factor (EF) enzymatic moieties to the cytosol. Antibodies against PA protect 42

against anthrax. We previously isolated toxin-neutralizing variable domains of camelid heavy-43

chain only antibodies (VHHs) and demonstrated their in vivo efficacy. In this work, gene 44

therapy with an adenoviral vector (Ad/VNA2-PA) promoting expression of a bispecific VHH-45

based neutralizing agent (VNA2-PA) consisting of two linked VHHs targeting different PA 46

neutralizing epitopes was tested in two inbred mouse strains, BALB/cJ and C57BL/6J, and found 47

to protect mice against anthrax toxin challenge and anthrax spore infection. Two weeks after a 48

single treatment with Ad/VNA2-PA, serum VNA2-PA levels remained above 1 µg/ml, with 49

some as high as 10 mg/ml. Levels were 10-100 fold higher and persisted longer in C57BL/6J 50

than in BALB/cJ mice. Mice were challenged with a lethal dose of LT or spores at various times 51

after Ad/VNA2-PA administration. The majority of BALB/cJ mice having serum VNA2-PA 52

levels >0.1 µg/ml survived LT challenge and 9 of 10 C57BL/6J mice with serum levels >1 µg/ml 53

survived spore challenge. Our findings demonstrate the potential for genetic delivery of VNAs as 54

an effective method for providing prophylactic protection from anthrax. We also extend prior 55

findings of mouse strain-based differences in transgene expression and persistence by adenoviral 56

vectors. 57

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Introduction 60

Bacillus anthracis produces two toxins which are responsible for allowing the bacterium to 61

establish disease and induce lethality in the host. Lethal toxin (LT) and edema toxin (ET) are 62

comprised of three proteins: protective antigen (PA), lethal factor (LF) and edema factor (EF). 63

PA is a receptor-binding component that transports LF (a protease) or EF (an adenylate cyclase) 64

into cells where they can manifest their catalytic activities through targeting of ubiquitous 65

substrates. EF targets ATP and converts it to cAMP, resulting in cellular dysfunction and 66

vascular events that can lead to lethality. LF cleaves the mitogen-activated protein kinase kinase 67

(MEK) family and rodent NLRP1 inflammasome sensors. LF plays an important role both early 68

and late in anthrax infection. Early in infection, inactivation of the MEK proteins by cleavage 69

leads to inhibition of a wide variety of innate immune cell responses which allows the bacterium 70

to evade the immune system, divide and disseminate. Cleavage of NLRP1 early in infection in 71

certain inbred rodents results in the activation of the inflammasome, macrophage pyroptosis and 72

induction of proinflammatory cytokines which induce a protective immune response. Thus, 73

certain inbred mouse strains are resistant to spore infection, while others are sensitive. Late in 74

infection, high levels of both anthrax toxins in the blood induce unknown vascular events which 75

contribute to the death of the host. The use of tissue-specific PA receptor knockout mice has 76

now identified target tissues for both toxins. While the mechanism of LT-induced death is 77

unknown, the cardiovascular system is clearly the important target and PA acts as the “gateway” 78

for all intoxication events (for a review of all preceding information, see (1)). 79

PA is an 83-kDa polypeptide that binds to receptors expressed in most tissues. It is then 80

cleaved by cell-surface proteases such as furin to a 63-kDa form that rapidly oligomerizes. 81

Heptamers or octamers of PA form binding sites for LF and EF (for review, see (1)). Because 82

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antibiotic treatment of B. anthracis infection is not effective after the anthrax toxins have 83

accumulated in the blood, the targeting of PA is an important therapeutic approach against the 84

disease. The majority of neutralizing antibodies against PA act on the receptor binding domain 4 85

and prevent the toxin interaction with cells. More rarely PA is neutralized through other 86

mechanisms (2). 87

Alpacas, camels and llamas are known to produce heavy chain only antibodies (for 88

review see (3, 4)). Variable domains of camelid heavy chain only antibodies (VHHs) can be 89

expressed as recombinant proteins which bind to antigen with similar affinity to the whole Ab, 90

but also have beneficial features which include resistance to high temperature and pH, as well as 91

the ability to access conformational epitopes in folded structures which are not generally reached 92

by conventional antibodies (3, 4). Our laboratories have established the efficacy of VHHs 93

against a variety of toxins (5-11). Linking of two or more neutralizing VHHs which target 94

different epitopes creates VHH-based neutralizing agents (VNAs), which have proven to be 95

much improved anti-toxin agents compared to a pool of their component monomers (8-10, 12). 96

We previously characterized a potent VNA for treatment of anthrax (VNA2-PA), made as a 97

heterodimer of two VHHs which neutralize PA by different mechanisms. One VHH, JKH-C7, 98

inhibits translocation of cell-surface generated PA63 oligomer, while the other, JIK-B8, is a 99

potent receptor blocker with a subnanomolar binding affinity for PA (6). 100

Gene therapy for in vivo expression of antibodies has had some success (13-17). In this 101

work we use a recombinant, replication-incompetent human adenovirus serotype 5 (Ad5) vector 102

that promotes expression and secretion into the serum of the VNA (Ad/VNA2-PA), thereby 103

passively “immunizing” the mice. We measured antibody (Ab) levels over an 8-week period 104

following a single bolus injection of the Ad/VNA2-PA. We performed studies in two different 105

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inbred strains in parallel, and found that robust protective Ab levels were rapidly established in 106

both strains, but at significantly different levels, and then dissipated at different rates. Challenge 107

studies done at various times post-treatment showed that mice having serum VNA levels above 1 108

µg/ml were protected from anthrax infection. Our results show the potential for VNA gene 109

therapy as an anthrax therapeutic. 110

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Materials and Methods 111

Ethics statement. Animal studies were done in accordance with protocols approved by the 112

Animal Care and Use Committees at NIAID (Protocols LPD8E, LPD9E). 113

Toxins. Endotoxin-free PA and LF were purified from B. anthracis as previously described (18). 114

LT is a combination of PA and LF, always used in equal amount. The LF used here is a 115

recombinant protein having an N-terminal sequence beginning HMAGG. LT concentrations 116

correspond to the concentration of each toxin protein (i.e., 100 µg/ml of LT is 100 µg/ml PA + 117

100 µg/ml LF). 118

Spores. Spores were prepared from the nonencapsulated, toxigenic B. anthracis Ames 35 (A35) 119

strain (19) by growth on NBY sporulation agar at 37 °C for 24 h followed by 5 days at room 120

temperature. Plates were inspected by microscopy to verify sporulation and spores gently 121

washed off with cold sterile water, followed by four additional cycles of sterile water washes and 122

centrifugation. Preparations were then heat-treated at 75°C for 1 h to kill any remaining 123

vegetative bacteria. Spore quantification was performed using a Petroff-Hausser counting 124

chamber (Hausser Scientific, Horsham, PA) and verified by dilution plating. 125

Ad/VNA2-PA construction and preparation. The generation of recombinant replication 126

incompetent Ad5-based vectors has been previously described (20). Briefly in a modification 127

from (7), pShCMV-JGf7 shuttle plasmid was used for subcloning the VNA2-PA coding 128

sequence (6), under control of the mammalian CMV promoter and followed by the bovine 129

growth hormone polyA signal. A control vector Ad/VNA-RT was created in a similar manner 130

with the sequence from two VHHs against ricin A chain (21). This control vector results in 131

secretion of a ricin reactive Ab with no binding to PA. Both shuttle plasmids were linearized and 132

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employed for homologous recombination with pAdEasy-1 plasmid and resultant plasmids 133

containing viral genomes were validated by PCR, restriction analyses and sequencing. The 134

plasmids were linearized with PacI to release the inverted terminal repeats of the viral genomic 135

DNA and transfected into 293 cells to rescue replication incompetent Ad/VNA2-PA and 136

Ad/VNA-RT. These Ad vectors were propagated in 911 cells, purified by centrifugation of CsCl 137

gradients, dialyzed and titers determined. 138

Adenovirus and monoclonal administration and bleeds. C57BL/6J or BALB/cJ mice (female, 8 139

weeks old) were obtained from Jackson Laboratories (Bar Harbor, Maine). Non-replicative 140

adenoviral vectors were diluted in sterile saline and injected by tail IV (100 µl/mouse, 3x1010 or 141

1.2 x1011 viral particles/study). In separate groups, BALB/cJ mice were injected with anti-PA 142

monoclonal 14B7 (100 µg or 10 µg/mouse, IV, 100 µl). Mice were bled by mandibular or tail 143

vein route at various days post adenoviral vector administration and serum separated using serum 144

separator tubes (Sarstedt, Newton, NC). 145

Serum VNA or monoclonal antibody measurement by ELISAs. Levels of the VNA2-PA or 146

monoclonal 14B7 in sera was measured by ELISA. Immulon II HB Immunoassay 96-well flat 147

bottom plates (Thermo Scientific, Franklin, MA) were coated with PA in PBS (10 µg/ml) 148

overnight at room temperature. Plates were washed with PBS and blocked with 100 µl/well of 149

1% Gelatin (Biorad, Hercules, CA) for 1 h. Sera from each mouse were serially diluted in 150

triplicate, incubated for 2-3 h, followed by removal and washing 3X with PBS-Tween (1XPBS+ 151

0.05% Tween 20). For sera from mice with adenoviral vector injections, an HRP-conjugated 152

anti-E-tag monoclonal antibody (mAb) (Bethyl laboratories, Montgomery, TX) was added to 153

each well at 1:3000 dilution and incubated for 2 h, followed by washing 5X with PBS-Tween. 154

For mice with 14B7 injected, a higher percentage of Tween 20 (1.3%) was used in washes and 155

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an HRP-conjugated anti-mouse secondary (Santa Cruz BT, Santa Cruz, CA) was used at 1:4000. 156

HRP substrate reagent (R&D systems, Minneapolis, MN) made of a combination of stabilized 157

hydrogen peroxide mixed with stabilized tetramethylbenzidine) was used for colorimetric 158

assessment of HRP activity by spectrometry (450 nM). Purified VNA2-PA or 14B7 dilutions 159

were used to construct standard curves and Ab concentrations were calculated relative to these 160

curves using GraphPad Prism software. 161

Toxin and Spore challenge. Mice were challenged with lethal doses of LT or spores at various 162

times following adenovirus administration. Toxin challenges were performed in BALB/cJ mice 163

which are known to be LT-sensitive, but are spore-resistant due to harboring an LT-responsive 164

Nlrp1b locus (22). Spore challenges were performed in the spore-sensitive C57BL/6J strain 165

which harbors a nonresponsive locus. LT (100 µg/mouse) was injected IP (500 µl) while spores 166

(5x107 A35 spores/mouse) were injected SC (200 µl) in the scruff of the neck. Mice were 167

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Results 169

We tested the efficacy of in vivo adenoviral production of heterodimeric anti-PA VNA2-PA by 170

injecting BALB/cJ (n=15) and C57BL/6J (n=15) mice with 3x1010 viral particles of Ad/VNA2-171

PA, and the same number of mice with a control adenoviral vector (Ad/VNA-RT) that produces 172

an anti-ricin A chain Ab not reactive to PA. The IV route was selected for administration of 173

vector, as earlier studies with a similar adenoviral vector expressing an anti-botulinum toxin 174

VNA showed 6- to 7-fold higher VNA levels following IV vs. IP injections, and >30-fold benefit 175

over the SC route (7). All mice were bled on day 10 and anti-PA VNA titers assessed. BALB/cJ 176

mice had anti-PA VNA concentrations ranging from 0.9 µg /ml – 2.3 mg/ml, with an average of 177

419 µg /ml, and a median of 35 µg/ml. These levels were far lower than the 6.2 mg/ml average 178

(7.03 mg/ml median) measured for C57BL/6J mice (Figure 1A). This is likely due to the fact 179

that BALB/c inbred mice eliminate cells containing the Ad transgene much more rapidly than do 180

C57BL/6 strain mice (23, 24). While 4 of 15 BALB/cJ mice had mg/ml levels of VNA, the rest 181

had 1-100 µg/ml. The lowest level of VNA in C57BL/6J mice was 1 mg/ml, with the majority 182

of mice having between 5-10 mg/ml of VNA (Figure 1A). Five mice from each strain were 183

challenged in a blinded fashion on day 11. BALB/cJ mice were challenged with 1xLD100 dose 184

of anthrax LT (100 µg, IP), while spore-sensitive C57BL/6J mice were challenged with 185

5xLD100 dose of 5x107 spores. All mice treated with the Ad/VNA2-PA survived, while all 186

challenged controls which had been treated with control vector succumbed (Figures 1B, 1C). 187

VNA2-PA serum levels were again assessed on day 18 for all mice and were found to be 188

reduced, but to very different levels in a mouse strain-dependent manner. In BALB/cJ mice, 189

11/15 had levels 10 µg/ml (Figure 2A), as will 190

be discussed later. Because protection against LT bolus challenge has historically required bolus 191

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administration of at least 50 µg of neutralizing mAb, we challenged the remaining BALB/cJ 192

mice (n=10), with 1xLD100 of LT on day 19. Of the ten challenged mice, 50% survived 193

challenge (Figure 2B). It appeared that mice with VNA levels of >1 µg/ml (>18.6 nM) survived, 194

with one exception, while levels

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challenged with anthrax spores (5xLD100 dose of 5x107/mouse) on day 34, there was a 215

substantial delay in onset of malaise and death in the mice treated with Ad/VNA2-PA, and 40% 216

of challenged mice survived (Figure 4B). Mice with 1 mg/ml (Figure 5). By 4 weeks, however, the levels of VNA had dropped 224

precipitously, to the 0.1- 1 µg/ml range for all mice, indicating that the relative net loss of VNA 225

in BALB/cJ mice occurred in proportion to the initial starting concentration, but at a similar pace 226

independent of vector dose. Not surprisingly, only 3 of 16 mice challenged with LT survived. 227

228

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229

Discussion: 230

We tested the ability of an Ad5-based adenoviral vector (Ad/VNA2-PA) expressing a bispecific 231

VHH-based neutralizing agent (VNA2-PA) consisting of two linked VHHs targeting different 232

anthrax toxin PA neutralizing epitopes to protect mice against anthrax toxin challenge and 233

anthrax spore infection. A single treatment with Ad/VNA2-PA, resulted in antibody levels as 234

high as 10 mg/ml. Levels were higher and persisted longer in C57BL/6J than in BALB/cJ mice. 235

LT-sensitive BALB/cJ mice having serum VNA2-PA levels >0.1 µg/ml survived LT challenge, 236

and spore-sensitive C57BL/6J mice with levels >1 µg/ml survived spore challenge. The studies 237

presented here indicate that adenoviral delivery of VHH-based neutralizing agents, VNAs, can 238

provide an excellent alternative to standard antibody therapeutics. Sustained levels of VNA with 239

a single administration of non-replicative Ad5 adenovirus allows the host to combat effects of 240

toxin or virulence factors for longer periods than repeated administration of purified antibody. 241

Furthermore, the gene therapy vectors can be used as prophylactic therapeutics if there is a 242

danger for exposure of large populations to toxic agents. The VHH-based therapeutics can also 243

be engineered to make VNAs which target multiple toxins or agents in a single product, 244

delivered with a single inoculation (9). These vectors also allow the possibility to deliver VNAs 245

to specific tissues sites through vector engineering (25). While Ad5 vector use in humans could 246

be limited by widespread pre-existing immunity, alternative gene therapy vectors such as 247

adenoviruses from simian sources or adeno-associated viruses are being developed that may 248

prove more practical for general use. 249

Interestingly, the levels of VNA in our current studies were not sustained for as long a 250

period as those observed for a similarly constructed anti-botulinum antitoxin VNA which was 251

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delivered at 3x1010, IV, into CD-1 Swiss mice (7). In that study, VNA levels remained at 1-10 252

mg/ml in half the mice even at 8 weeks post inoculation, although some mice had significantly 253

lower levels as early as 10 days post inoculation. Thus, the range of serum VNA could vary 254

from 0.01 ng/ml to >100 µg/ml in the mice at 6-8 weeks after vector administration. The reason 255

for this is very likely the genetic heterogeneity of CD-1 mice, which are outbred. Genetic factors 256

have been previously reported to influence the efficiency of Ad infection and/or transgene 257

production (24, 26, 27) and it is not surprising that these factors would be more variable in the 258

outbred mice than observed within the two inbred lines used in the current study. The same 259

genetic factors are expected to exist among humans and thus lead to differential responses to 260

various forms of antibody and VNA gene therapy. 261

Our findings that protection against anthrax toxin is possible for weeks after a single 262

administration of Ad/VNA2-PA suggest that adenoviral anti-anthrax therapeutics are a viable 263

option as a future therapeutic agent for this disease. The options of administering intranasal 264

Ad/VNA2-PA vectors, or parenterally administering Ad/VNA2-PA vectors designed to promote 265

pulmonary VNA expression (28), may result in even more effective therapeutics for anthrax 266

exposures. Future studies will focus on tissue-specific targeting of VNA gene therapy vehicles 267

for more efficient neutralization of toxic effects at relevant disease sites. 268

269

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270

Acknowledgements 271

This research was supported in part by the Intramural Research Program of the National Institute 272

of Allergy and Infectious Diseases (NIAID) and by National Institutes of Health Grant U54 273

AI057159 (CBS). The authors thank the staff of the NIAID Comparative Medicine Branch for 274

assistance with vivarium studies 275

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Figure Legends 278

279

Figure 1. Day 10 sera analyses and Day 11 challenge studies. A) Groups of BALB/cJ or 280

C57BL/6J mice (n=15/group) were injected with Ad/VNA2-PA or Ad/VNA-RT (3x1010 viral 281

particles), and VNA2-PA levels were assessed on day 10. Each circle refers to a single mouse, 282

with the upper panel showing Ad/VNA2-PA groups and lower panel showing the control vector 283

groups. The dashed lines indicate the average (black) and median (red) Ad/VNA2-PA levels for 284

each strain. The open boxes indicate the mice that were challenged, with results shown in panels 285

B and C. B) BALB/cJ mice treated with Ad/VNA2-PA and control mice (n=5/see panel A) were 286

challenged on day 11 with LT (100 µg, IP) and monitored for malaise and survival. C) 287

C57BL/6J mice treated with Ad/VNA2-PA and control mice (n=5/group, see panel A) were 288

challenged with anthrax A35 spores (5x107/mouse) and monitored for malaise and survival. 289

290

Figure 2. Day 18 sera analyses and Day 19 challenge study results. A) Day 18 sera from the 291

mice that survived challenge as well unchallenged mice described in Figure 1 were analyzed for 292

VNA2-PA levels. Each circle refers to a single mouse, with the upper panel showing Ad/VNA2-293

PA groups and lower panel the control vector groups. The open boxes indicate the two groups of 294

n=10 BALB/cJ mice that were challenged, with results shown in panel B. The black filled 295

rectangles indicate the absence of sera for mice that succumbed in previous challenge. The 296

dashed lines indicate the average (black) and median (red) Ad/VNA2-PA levels for each strain. 297

The “D” identifies 4 mice that succumbed to the challenge described in panel B. B) BALB/cJ 298

mice treated with Ad/VNA2-PA and control mice (n=10/group, see panel A) were challenged on 299

day 19 with LT (100 µg, IP) and monitored for malaise and survival. 300

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301

Figure 3. 14B7 levels in sera and LT challenge outcome. A) BALB/cJ mice (n=5/group) were 302

injected with either 10 or 100 µg of mAb 14B7 (IV, 100 µl) and bled at 2 h to assess circulating 303

levels of PA-specific monoclonal. The solid line indicates the average mAb serum level. B) All 304

mice from panel A were challenged with LT (100 µg, IP), 2.5 h following mAb administration. 305

Mice were monitored for a week for signs of malaise. 306

307

Figure 4. Day 25 and 33 sera analyses and Day 34 spore challenge study results. A) A 308

comparison of day 18 sera VNA2-PA levels from Figure 2 is shown with levels on day 25 and 309

33. The dashed line indicates the average Ad/VNA2-PA levels for the ten C57BL/6J mice on 310

DAY 33. The open box indicates the mice that were challenged on day 34. The “D” identifies 6 311

mice that succumbed to the challenge described in panel B. B) C57BL/6J mice treated with 312

Ad/VNA2-PA and control mice (n=10/group, see panel A) were challenged on day 34 with 313

anthrax A35 spores (5x107/mouse) and monitored for malaise and survival. 314

315

Figure 5. Study 2: Week 2 and Week 4 sera analyses and Week 5 LT challenge studies. A) 316

BALB/cJ mice (n=16/ group) were injected with Ad/VNA2-PA or Ad/VNA-RT (3x1010 viral 317

particles), and VNA2-PA levels were assessed at 2 weeks and 4 weeks. Results shown are for 318

the Ad/VNA2-PA group, as Ad/VNA-RT mice never had PA-specific antibodies. The dashed 319

lines indicates the average Ad/VNA2-PA levels at 2 and 4 weeks. Each circle refers to a single 320

mouse. “S” identifies 3 mice that survived the challenge shown in B. B) All mice in panel A 321

were challenged at 5 weeks with LT (100 µg, IP) and monitored for malaise and survival. 322

323

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