aac accepts, published online ahead of print on 19 january...

IMBODEN et al. Fusion proteins effective against C. parvum

1

Antibodies fused to innate immune molecules reduce initiation of Cryptosporidium 1

parvum infection in mice. 2

3

4

Running title: Fusion proteins effective against C. parvum 5

6

7

Michael Imboden 1*

, Michael W. Riggs2, Deborah A. Schaefer

2, E. Jane Homan

1, Robert 8

D. Bremel1 9

10

1. ioGenetics LLC 11

2. Department of Veterinary Science and Microbiology, University of Arizona 12

* Corresponding Author, ioGenetics LLC, 3591 Anderson Street, Madison, WI 53704, 13

608 310 9540, fax 608 310 9544, [email protected] 14

15

Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.00754-09 AAC Accepts, published online ahead of print on 19 January 2010

on April 21, 2018 by guest

http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


2

Abstract. 1

At the current time there are no completely effective treatments available for 2

Cryptosporidium parvum infections in humans and livestock. Based on previous data 3

showing the neutralizing potential of a panel of monoclonal antibodies developed against 4

C. parvum, and innate immune peptides and enzymes having anticryptosporidial activity, 5

we engineered several of these antibodies into antibody-biocide fusions. We 6

hypothesized that the combination of high affinity antibody targeting with innate immune 7

molecule-mediated killing would result in a highly effective new antiprotozoal agent. To 8

test this we expressed antibody-biocide fusions in a mammalian cell culture system and 9

used the resulting products for in vitro and in vivo efficacy experiments. Antibody-10

biocide fusions efficiently bound to, and destroyed, C. parvum sporozoites in vitro 11

through a membrane-disruptive mechanism. When antibody-biocide fusions were orally 12

administered to neonatal mice in a prophylactic model of cryptosporidiosis, the induction 13

of infection was reduced by up to 81% in the mucosal epithelium of the gut based on 14

histopathological scoring of infectious stages. Several versions of antibody fusions that 15

differed in antigen specificity and biocide induced a strong inhibitory effect on initiation 16

of infection. The results lay the groundwork for the development of a new class of 17

antimicrobials effective against Cryptosporidium. 18

19

20

Introduction 21

Cryptosporidium parvum is a zoonotic apicomplexan parasite which primarily infects 22

cattle (10). C. parvum causes an economically important diarrheal disease in calves and 23


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


3

other livestock especially in the first few weeks of life (8). At least two species of 1

Cryptosporidium commonly infect man; C. parvum (previously known as C. parvum type 2

2) and the human-specific C. hominis (previously known as C. parvum type 1) (34). In 3

otherwise healthy human hosts the diarrhea cryptosporidiosis produces is rarely fatal, but 4

deaths may occur in immunocompromised individuals (6,13,17,29). In tropical countries 5

diarrhea resulting from Cryptosporidium spp. infection may result in chronically 6

impaired childhood development (9,14). 7

No consistently or completely effective therapeutic drugs exist for cryptosporidiosis in 8

man or livestock. Currently management depends on supportive therapy and good 9

hygiene (28). Nitazoxanide has been approved for use in humans, but Cryptosporidium 10

lacks the specific enzyme target for this drug and results are mixed (36). Recent trials of 11

nitazoxanide in calves for prevention or treatment of cryptosporidiosis have likewise 12

proved disappointing, with no clinical or parasitologic efficacy observed (26). New 13

approaches to therapy for cryptosporidiosis are therefore urgently needed (32). 14

Riggs et al developed a monoclonal antibody (MAb) 3E2, which binds to the 15

circumsporozoite-like antigen (CSL), and showed that it could partially neutralize 16

Cryptosporidium parvum in mice (24) and calves (19), albeit at pharmacologically high 17

dosages. Nevertheless, the high cost of production of this antibody from the hybridoma 18

renders its use in the field impracticable. Schaefer et al then developed a library of 19

monoclonal antibodies against functionally defined C. parvum sporozoite antigens and 20

showed some to be effective at reducing infection in mice (25). A formulation of MAbs 21

targeting three different neutralization-sensitive antigens provided significant additive 22


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


4

efficacy over that of the individual MAbs, or combinations of two MAbs, when 1

administered orally to mice. 2

Several workers have shown that antimicrobial peptides and certain enzymes are active 3

against apicomplexan parasites (1,11,16,30,31,35). In general, high doses were needed to 4

effect neutralization. In a prior study we explored the effect on in vitro viability of C. 5

parvum of a number of antimicrobial peptides and enzymes (7). 6

Based on these demonstrated effects of peptides and antibodies against parasites, we 7

hypothesized that by using the high affinity binding of antibodies we could target 8

antimicrobial peptides and enzymes (herein collectively termed “biocides”) for delivery 9

at the protozoal surface. We reasoned that this precision targeting would reduce the 10

dosage needed and, therefore, cost and potential host toxicity. 11

To do this we needed to construct fusion proteins, comprising the variable and constant 12

regions of antibodies that specifically bind to C. parvum with selected peptide biocides, 13

in such a way that the function of both parts was retained. We used the previously 14

developed library of hybridomas as a source of immunoglobulin variable regions directed 15

to a variety of epitopes on C. parvum sporozoites, and selected antimicrobial 16

peptides/enzymes as a source of biocides (7). By using genetic engineering to combine 17

these elements, and a retrovector gene transfer system, we were able to express functional 18

fusion proteins at high yield in mammalian cell culture. In addition, we modified the 19

isotype and configuration of selected immunoglobulin molecules to change size and 20

functionality. We then tested these fusion proteins for their efficacy in killing C. parvum 21

infectivity in vitro and in reducing infection in neonatal mice that were concurrently 22

challenged with C. parvum oocysts. Using a mouse model, this study demonstrates that 23


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


5

significantly greater neutralization of C. parvum can be obtained using the fusion proteins 1

compared to antibody or biocide alone and offers a promising alternative approach to 2

control of C. parvum. 3

Materials and Methods. 4

Hybridomas 5

Three hybridomas producing antibodies directed to different neutralization-sensitive 6

antigens on Cryptosporidium parvum were previously created (21,22,24,25) (Table 1). 7

Since MAb 3E2 has been shown earlier to have significant neutralizing efficacy against 8

C. parvum infection in neonatal mice (24) and calves (19), we included it here as a 9

positive control. As an isotype control antibody we used MAb 166 (kind gift of Dr. K. 10

Ziegler, Emory University, GA), directed to Listeria monocytogenes (37). Hybridoma-11

derived and recombinant MAb 166 do not bind to C. parvum sporozoites as determined 12

by immunofluorescence assay (IFA) (data not shown). 13

Assembly of genetic constructs 14

Total RNA was isolated from hybridoma cells (RNeasy kit Qiagen Inc. Valencia, CA) 15

and reverse transcribed into cDNA using oligo dT primers (AffinityScript cDNA 16

synthesis kit, Stratagene, La Jolla, CA). Immunoglobulin variable region genes were 17

amplified from cDNA using degenerate upper primers semi-specific for the signal 18

peptide region combined with lower primers specific for the constant region (Novagen 19

Ig-primer set, EMD Biosciences, San Diego, CA) and the PCR products obtained were 20

cloned (Strataclone PCR cloning kit, Stratagene, La Jolla, CA). Multiple clones derived 21

from the same PCR product were sequenced to test for PCR-derived mutations and 22


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


6

correct reading frame using Lasergene (DNAstar Inc., Madison, WI) and compared with 1

sequences in Genbank to confirm that they were of mouse immunoglobulin origin. 2

Immunoglobulin gene constant regions were extracted from hybridoma cDNA using 3

primers (oligonucleotides obtained from Integrated DNA Technologies, Coralville, IA) to 4

the known constant sequence of either murine IgG1 or IgG2b isotype. The (G4S)3 linker, 5

including flanking regions compatible with heavy chain sequence at the 5’-end and 6

biocide sequence at the 3’-end, was synthesized by Blue Heron Biotechnology (Bothell, 7

WA). The gene for human phospholipase A2 group IIA (PLA2) was obtained from the 8

ATCC gene collection (MGC-14516). The coding region for LL37, the active portion of 9

human cathelicidin hCAP-18 was assembled by PCR amplification of three long 10

overlapping oligomers that were based on Genbank NM_004345. The RNA export and 11

stabilization element (RESE) is based on the woodchuck hepatitis virus RNA export 12

element and enhances RNA export from the nucleus in the absence of RNA splicing (38). 13

To engineer the various IgM-based constructs we first isolated the variable and constant 14

regions from the 3E2 hybridoma cell line as described above and constructed a full size 15

IgM molecule for testing purposes. In the absence of the J-chain, IgM spontaneously 16

forms hexamers which we confirmed by size analysis using polyacrylamide gel 17

electrophoresis (PAGE). Once we confirmed binding of the 3E2 hexamer to sporozoites 18

by IFA (described below), the 3E2 sequences were used to construct monomeric and 19

halfmeric fusion proteins. This was achieved by eliminating two or three of the interchain 20

disulfide bonds in the IgM heavy chain genes. This was done as described by Wiersma et 21

al (33) using site-directed mutagenesis PCR to introduce the requisite cysteine to serine 22


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


7

codon changes of the nucleotide sequence C337S+C414S+C575S to make halfmers and 1

C414S+C475S to make monomers. 2

All elements were assembled in a series of overlap PCR reactions and the final product 3

containing flanking restriction endonuclease sites was cloned into a murine leukemia 4

virus (MLV) based replication incompetent retroviral expression system (Pantropic 5

Retroviral Vector System, Clontech, Mountain View, CA) modified to include the simian 6

cytomegalovirus (CMV) promoter (GenBank Accession U38308) (Figure 1). Due to the 7

very high gene transfer rates into mammalian host cells achieved with the retroviral 8

system, the Neor gene for selection is not essential and was removed from the retrovector 9

backbone. 10

To confirm the production of correctly assembled recombinant antibody biocide fusion 11

products, PAGE was performed under both reducing and non-reducing conditions, 12

followed by Western blotting using an affinity purified goat anti-mouse IgG antibody or 13

anti-mouse IgM antibody (Bethyl Laboratories, Montgomery, TX). 14

Expression in cell culture 15

The retroviral construct containing the gene of interest was co-transfected with plasmid 16

containing the gene for vesicular stomatitis glycoprotein into GP2-293 packaging cells 17

(Pantropic Retroviral Expression system, Clontech, Mountain View, CA) to produce 18

infectious replication-incompetent pseudotyped retrovector particles. These were 19

harvested by centrifugation (75,000 × g) and resuspended for 2 h, then used to transduce 20

CHO cells. Vector was removed and replaced with fresh SFM4 (Hyclone, Logan, UT) 21

medium after 16 h. Ten to twelve days after transduction cell pools were analyzed by 22

ELISA for the detection of recombinant products using a heavy chain capture and light 23


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


8

chain signal generation (Bethyl Laboratories, Montgomery, TX). Upon confirmation of 1

presence of correctly assembled immunoglobulins, individual cells were isolated in 96 2

well plates by limiting dilution. After 12 days, supernatants were re-analyzed and the 3

highest producing clones were selected and expanded. Recombinant products were 4

produced in standard tissue culture flasks or 500 ml Erlenmeyer flasks with agitation. 5

Typically, cultures were harvested after 8-10 days of incubation, cells removed by 6

double-centrifugation (400 × g for 10 min, 6000 × g for 10 min), and supernatants 7

analyzed to determine product concentration using ELISA co-detection of 8

immunoglobulin heavy and light chain. Recombinant products or hybridoma-derived 9

MAbs used for these studies were either prepared from unprocessed cell culture 10

supernatants or supernatants concentrated up to 3 fold using Amicon Centricon Plus-20 11

(Millipore, Billerica, MA) to provide equal protein concentrations. 12

The recombinant products expressed are described in this paper using the following 13

nomenclature: variable region source-recombinant isotype-biocide, for instance 4H9-G1-14

LL37. Recombinant products lacking a biocide fusion are described as variable region 15

source-recombinant isotype, for instance 4H9-G1. Hybridoma derived MAbs are 16

described as hybridoma name MAb, for instance 3E2 MAb. 17

Cryptosporidium parvum oocyst source 18

The Iowa C. parvum isolate (12) has been maintained since 1988 by propagation in 19

newborn Cryptosporidium-free Holstein bull calves (21,23) which were the source of 20

oocysts for all experiments. Oocysts were isolated from calf feces by sucrose density 21

gradient centrifugation and stored in 2.5% KCr2O7 (4°C) (2,23). For challenge of 22

neonatal mice, oocysts were used within 30 days of isolation and disinfected with 1% 23


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


9

peracetic acid immediately prior to administration (20). To obtain isolated sporozoites for 1

use in vitro, oocysts were hypochlorite-treated prior to excystation, (37°C, 0.15% 2

[wt/vol] taurocholate, 1 h), then passed through a sterile polycarbonate filter (2.0 µm pore 3

size; Poretics, Livermore, CA) and used immediately (23,25). Oocyst excystation was 4

determined immediately prior to mouse administration, or to obtain isolated sporozoites, 5

and always exceeded 90%. 6

Assays for binding of recombinant products to sporozoites and in vitro assessment of 7

viability. 8

For immunofluorescence assays to assess binding, excysted sporozoites were aliquoted 9

onto Teflon-coated multiwell glass slides, air-dried, and then gently heat fixed. 10

Individual wells were incubated (30 min, 37°C) with concentration-matched recombinant 11

fusion products, recombinant antibody, isotype-matched control MAb of irrelevant 12

specificity, or CHO cell supernatant control, washed with PBS, incubated with 13

fluorescein-conjugated affinity-purified goat anti-mouse IgM/IgG/IgA (Kirkegaard & 14

Perry, Gaithersburg, Md.), washed, and then examined by epifluorescence microscopy. 15

To quantify parasiticidal activity of recombinant products, sporozoite viability after in 16

vitro incubation with individual products was assessed using fluorescein diacetate (FDA) 17

and propidium iodide (PI) as previously described (3,7). In brief, freshly excysted 18

sporozoites were incubated (15 min, 37°C) in CHO medium containing individual 19

recombinant products (50 µg/ml) or spent CHO cell medium (n = 3). Heat killed (20 sec, 20

100°C) sporozoites were used as an internal control. FDA (8 µg/ml final concentration) 21

and PI (3 µg/ml final concentration) were added to the sporozoite preparations, incubated 22

further (30 min, 21°C), then examined in fluid phase wet mounts by epifluorescence 23


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


10

microscopy. A minimum of 100 sporozoites were counted for each preparation to 1

determine the percent reduction in viability [(CHO-treated sporozoite mean viability - 2

recombinant product-treated sporozoite mean viability) ÷ CHO-treated sporozoite mean 3

viability] × 100. The mean values for test and control preparations were examined for 4

significant differences using JMP software and ANOVA analysis of variance (SAS, Cary, 5

NC). 6

Evaluation of recombinant products for efficacy in vivo 7

Groups of 10 eight-day-old SPF ICR mice were administered, by gastric intubation, 5 × 8

104 oocysts (50 × MID50) (23) concurrently with recombinant antibody fusions or 9

combinations of individual MAbs and biocides (100 µl, concentration range 10-100 10

µg/ml). At 3 and every 12 h thereafter, mice received additional treatments (100 µl, 11

concentration range 10-100 µg/ml) by gastric intubation for a total of nine treatments. 12

Cimetidine (10 mg/kg) was included with all treatments. Groups of 10 eight-day-old 13

control mice were treated identically with CHO cell culture supernatant or recombinant 14

antibody alone. After euthanasia at 92-94 h p.i., the jejunum, ileum, cecum, and colon were 15

collected, coded, and examined histologically by the same investigator, without knowledge 16

of treatment group, for C. parvum stages in mucosal epithelium. Scores of 0, 1, 2 or 3 (0, no 17

infection; 1, < 33% of mucosa infected; 2, 33 to 66% of mucosa infected; and 3, > 66% of 18

mucosa infected) were assigned to longitudinal sections representing the entire length of (i) 19

terminal jejunum, (ii) ileum, (iii) cecum, and (iv) colon, then summed to obtain an infection 20

score (0 to 12) for each mouse (21,24). Percent reduction of infection was calculated as 21


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


11

[(Control mean infection score - product mean infection score) ÷ Control mean infection 1

score × 100]. The control treatment in all in vivo experiments was CHO cell culture 2

supernatant. 3

Experimental results were analyzed in JMP version 8 (SAS Institute Cary, NC) by 4

ANOVA. Differences between means were tested with a Tukey-Kramer HSD with alpha 5

= 0.05. 6

All mice were maintained in BSL2 biocontainment at the University of Arizona and in 7

accordance with the PHS Guide for the Care and Use of Laboratory Animals. 8

Accession Numbers 9

Sequences coding for the variable regions of MAb 18.44, 4H9 and 3E2 were deposited in 10

Genbank under the accession numbers 1._____ 11

Results. 12

Production of recombinant antibodies and antibody-biocide fusions 13

Recombinant protein fusions comprising monoclonal antibodies and biocides were 14

assembled using the basic retroviral constructs shown in Figure 1. Use of the retroviral 15

system allowed us to generate stable cell lines for all the recombinant products shown in 16

Table 1 in a short period of time. Cell supernatant-containing products were tested in the 17

in vitro assay and the neonatal mouse model. All products showed the expected sizes on 18

Western blots for either heavy chains alone, heavy chain-biocide fusions or kappa light 19

chains (data not shown). Binding specificity of the recombinant 4H9, 3E2 and 18.44–20

1 Genbank Accession numbers will be added at modification stage


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


12

derived products for C. parvum sporozoites, and lack of binding of recombinant 166-1

derived products, was confirmed by immunofluorescence assay (data not shown). Table 1 2

shows the recombinant products and control antibodies tested in this study. These 3

products were generated consecutively. To conserve resources and prevent redundancy 4

not every possible combination of isotype and biocide was produced a priori, rather, the 5

decisions as to which combinations to produce were based on data obtained from prior 6

fusions. 7

Direct effects of antibody biocide fusion proteins on sporozoite viability 8

An in vitro viability assay was performed using various different versions of antibody-9

biocide fusions comprising the 3E2 (anti-CSL), 4H9 (anti-GP25-200) and 18.44 (anti 10

CPS-500) specificitiescombined with the LL37 and PLA2 biocides (Fig. 2). This 11

assessment showed that antibody-biocide fusions targeting any one of these three 12

different antigens on the sporozoite surface mediate significantly higher efficacy at 13

killing sporozoites in vitro than their stand-alone antibody counterparts. Since the IgM-14

based fusion 3E2-M-LL37, which adopts a hexamer configuration, presented technical 15

difficulties for expression and purification, we designed size-reduced versions comprising 16

an IgM monomer (two heavy chains + two light chains), and IgM halfmer (one heavy 17

chain + one light chain) fused to LL37. The 3E2-based fusions showed significantly 18

increased efficacy at killing sporozoite in vitro when compared to the hybridoma-derived 19

3E2 MAb (Fig. 2A). 20

Moreover, we also wanted to test if an isotype switch from IgM to IgG1 would maintain 21

or enhance the efficacy of the 3E2 fusions. Surprisingly, the 3E2-G1-LL37 fusion 22

showed outstanding efficacy in vitro. Unfortunately this construct did not express well as 23


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


13

a recombinant molecule and we were unable to produce quantities sufficient to perform 1

in vivo testing. We subjected the 4H9-G1-LL37 to long-term storage at 4°C over a period 2

of 3 months to evaluate stability. Storage resulted in a loss of activity of only 2.6%, 3

indicating good stability under refrigeration temperatures. The 4H9-G1-PLA2 fusion was 4

also tested in this series but did not show any direct effect on the viability of C. parvum 5

sporozoites in vitro (data not shown). 6

Figure 2B shows the effects on sporozoite viability of 18.44 MAb, alone and in 7

combination with PLA2 or LL37, compared to the corresponding 18.44 MAb-biocide 8

fusions. The 18.44-G3 MAb itself exhibited a low rate of sporozoite killing, which 9

increased slightly by simultaneous application of the MAb with recombinant PLA2 as 10

individual, non-fused molecules. However, when 18.44 was expressed as an IgG1 with 11

PLA2 as a C-terminal fusion, the resulting18.44-G1-PLA2 fusion protein showed an 12

approximately 3.5 fold increase in activity over that of 18.44 and PLA2 tested in 13

combination. The 51% reduction in sporozoite viability achieved by 18.44-G1-PLA2 is 14

one of the strongest we have detected in the in vitro assay. The 18.44-G1-LL37 fusion 15

protein exhibited a lower activity than the 18.44-G1-PLA2 fusion indicating that a given 16

epitope on the surface of C. parvum may differ in its suitability to mediate targeted 17

delivery of different biocides. The outcome of this experiment provided the initial 18

indication that antibody biocide fusions have a direct and greater impact on sporozoite 19

survival in vitro compared to the corresponding stand-alone antibodies. 20

To evaluate morphological effects, sporozoites were exposed to various fusion proteins 21

for 30 minutes and then analyzed by immunofluorescence. Figure 3A shows a 22

representative result of sporozoites exposed to either CHO cell supernatant, monoclonal 23


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


14

antibodies or the 4H9-G1-PLA2 fusion, none of which showed any direct killing activity. 1

Figure 3B is representative for the activity shown by all LL37 fusions tested. The 2

observation of spherical shapes and increased number of dead cells are indicative of the 3

degenerative process associated with disturbance of the osmoregulatory system in the 4

sporozoite. The killing rate of sporozoites by the fusion proteins varied considerably 5

depending on components used. . 6

In anticipation of oral administration of the fusion protein products, we examined the 7

effect of pH on the stability of 4H9-G1-LL37. After incubation at various pHs (PBS at 8

pH 2, 3, 4, 5, 7) at 37°C for 90 min, the fusion protein samples were neutralized to pH 7 9

and tested in vitro for their direct effect on sporozoite viability. There was no decrease in 10

effectiveness of the fusion protein following low pH treatment (Figure 4). Interestingly, 11

the efficacy of 4H9-G1-LL37 almost doubled after exposure at pH 2. As our goal in this 12

experiment was simply to determine whether pH values to which the fusion proteins 13

would be exposed during gastrointestinal transit would be deleterious, the chemical 14

mechanism for the observed enhancement of activity upon low pH exposure is the subject 15

of ongoing studies. 16

Fusion proteins inhibit initiation of C. parvum infection in neonatal mice. 17

Cell culture-derived supernatants containing recombinant fusion proteins 4H9-G1-LL37, 18

4H9-G2b-LL37 and 18.44-G1-PLA2, and control 3E2 MAb were orally administered to 19

neonatal mice concomitantly with the oocyst-challenge. Increasing dosages of fusion 20

proteins lead to a greater prophylactic effect on infection as determined by intestinal 21

section scoring to quantify intracellular C. parvum stages (Figure 5). The dose response 22

patterns of 4H9 fusion proteins in both the IgG1 and IgG2b formats were similar. A 23


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


15

similar dose response pattern was also observed with the 18.44-G1-PLA2 fusion protein. 1

The efficacy of all three fusions leveled off as the dose was increased to 7.7 mg/kg/day. 2

The CHO cell supernatant (spent medium) controls had no significant effect on infection 3

levels (data not shown). MAb 3E2 IgM pentamer positive control had to be used at a 4

dosage of 462 mg/kg/day to induce a reduction of initiation of infection of 37% (lower 5

dosages resulted in insignificant reduction of initiation of infection – data not shown). No 6

adverse effects of the treatments were observed based on clinical appearance, growth and 7

suckling response. Further, no evidence of host toxicity was observed based on 8

histopathological evaluation of intestinal sections for morphologic changes. 9

The above data clearly show that multiple recombinant fusion proteins differing either in 10

their binding specificity or in their biocide component, can be highly efficacious at 11

reducing C. parvum infection in neonatal mice. Furthermore, the 4H9 and 18.44 fusions 12

have significantly greater neutralizing activity than the hybridoma antibody 3E2 used as a 13

comparator, showing efficacy at doses that are 60-fold lower. 14

Antibody-biocide fusions are more effective than the sum of their parts at reducing 15

initiation of infection in the neonatal mouse model 16

In vitro data presented in Figure 2B showed that selected antibody biocide fusions are 17

significantly more effective at killing sporozoites than an equimolar matched mixture of 18

single antibody and single biocide molecules. To further extend these observations, we 19

compared the efficacy of antibody plus biocide administered as separate molecules with 20

that of a fusion of the two molecules against a C. parvum oocyst challenge in neonatal 21

mice. The effectiveness of the fusion molecules was significantly greater than that of 22

individual MAb and biocide components given as separate molecules at equimolar 23


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


16

amounts (Table 2). The 4H9-biocide fusion molecules reduced intestinal infection by 74-1

81%, whereas the reduction achieved with a combination of 4H9-G1 plus PLA2 was 2

31%, and with 4H9-G1 plus LL37 was 23%. The 4H9-G1-LL37, 4H9-G2b-LL37, and 3

4H9-G1-PLA2 fusions were approximately equivalent in efficacy. 4

Antibody biocide fusions directed to different epitopes have a synergistic effect 5

Various fusion proteins were created using the variable region of anti-CSL MAb 3E2 6

(Table 1). 3E2 IgM monomer and 3E2 IgM halfmer fusions with LL37 were compared to 7

the 4H9-biocide fusion molecules in the neonatal mouse model. When the 3E2-M 8

monomer-LL37 was given to mice at a dose of 7.7 mg/kg/day, a significant reduction of 9

initiation of infection over control treatment was observed, demonstrating that the activity 10

of antibody fusion proteins in vivo can be mediated through different surface exposed 11

epitopes. This observation further broadens the number of potential targets on the 12

sporozoite surface for fusion protein-mediated neutralization. The most effective 13

molecule of the series was the 3E2-Mhalfmer-LL37 fusion which reduced infection by 14

82% at a relatively low dose. We have previously observed that combinations of MAbs 15

targeting different neutralization-sensitive antigens can provide significant additive 16

efficacy over that of the individual MAbs (25). Therefore we next evaluated the effect of 17

a combined fusion protein treatment targeting two distinct epitopes. When 4H9-G1-LL37 18

and 3E2-Mmono-LL37 were given together, each at half the dose used individually (3.8 19

mg/kg/d), a significant increase in efficacy over 3E2-Mmono-LL37 used alone at the 7.7 20

mg/kg/d dose was observed indicating a synergistic effect (Table 3). 21


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


17

Discussion 1

In an effort to address the urgent need for therapeutics effective against Cryptosporidium 2

parvum we created a number of recombinant antibody-peptide and antibody-enzyme 3

fusion proteins and evaluated their efficacy in both in vitro and in vivo assays. These 4

recombinant molecules were constructed using genes derived from the hybridoma 5

libraries developed by Riggs and Schaefer et al (21,24,25) and genes for human secretory 6

phospholipase IIA and human cathelicidin antimicrobial peptide LL37. Using a retroviral 7

expression system without the need for selectable markers, CHO production cell lines 8

were created for each antibody and antibody-fusion protein tested. Overall, the 9

expression levels of the fusion proteins in the CHO system were comparable to the 10

monoclonal antibodies indicating that these molecules offer promise as novel therapeutic 11

entities in that they can be produced effectively in established expression systems. The 12

recombinant proteins were tested as concentrated cell culture supernatants for their ability 13

to kill sporozoites in vitro and to reduce initiation of C. parvum infection in a neonatal 14

mouse model. 15

When sporozoites were exposed directly to recombinant antibodies lacking the biocide 16

fusion, only a minimal effect on viability was observed. Conversely, when the same 17

recombinant antibody was fused to LL37, a strong anti-sporozoite effect was seen, 18

leading to a rapid loss of osmotic equilibrium and viability of the sporozoites within 19

minutes. Similarly, fusion between 18.44 and PLA2 showed significantly enhanced 20

activity over antibody and PLA2 used as separate molecules in vitro. 21

Exposure of 4H9-G1-LL37 to low pH did not reduce the activity of the fusion protein; 22

rather it increased the anti-sporozoite activity. A possible explanation may lay in the 23


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


18

observation of Johannson et al (15) who have shown that at low pH (2-3) the LL37 1

‘uncoils’ which could potentially release inhibiting components bound to it. The same 2

workers also showed that upon neutralization of the pH, the LL37 regains its α-helical 3

conformation. In other animal studies, now in progress, we have observed that antibody-4

LL37 peptide fusions are resistant to degradation during passage through the stomach and 5

maintain binding specificity in the absence of antacid treatment (data not shown), which 6

is consistent with the data presented in Figure 4. 7

When administered orally to neonatal mice, coincident with a C. parvum oocyst 8

challenge, the fusion proteins showed a dose dependent response in their ability to 9

prophylactically reduce infection. The fusion products 4H9-G1-LL37, 4H9-G2b-LL37 10

and 4H9-G1-PLA2 each had an effect equivalent to 3E2 MAb, but when given to mice at 11

a 60 fold lower dosage than 3E2. Heretofore, 3E2 MAb has been considered the 12

neutralizing monoclonal antibody standard for comparison for passive immunization 13

against C. parvum (24). When selected antibodies and biocides are administered as single 14

molecules, each at a concentration equimolar to the corresponding fusion proteins, they 15

have a significantly decreased neutralizing effect from that of the fusion. Binding of the 16

high affinity antibody allows delivery of a low, but effective, dose of the antimicrobial 17

peptide LL37 or the PLA2 enzyme to the parasite surface. 18

Overall our in vivo mouse model data show a stronger effect of antibody-biocide fusions 19

on C. parvum infection (up to 82% prophylactic reduction of intestinal cell infection) 20

than the in vitro viability assay (up to 50% sporozoites killing). Since this has been seen 21

consistently in a large number of experiments outside the scope of this paper, it is quite 22

possible that "killing" in the in vitro assay is underestimating the actual damage that the 23


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


19

molecules have done to the organism.. It is possible that neutrophils, monocytes and 1

macrophages are stimulated through interaction with the Fc portion of the 2

immunoglobulin and potentially by the LL37 peptide (5,27) in immunologically sensitive 3

locations such as Peyer’s patches. Such interactions could lead to a more generalized 4

immune stimulation that might beneficially impact clearance of sporozoites thus 5

complementing the direct killing effect of the fusion proteins. 6

The most active anticryptosporidial fusion we examined was 3E2-Mhalf-LL37, which 7

comprises only one IgM heavy chain fused to LL37 and one light chain. The smaller size 8

of this molecule (MW 94.7 kDa) may allow greater access to the known repetitive CSL 9

epitope recognized by 3E2 MAb and thereby facilitate interaction of LL37 with the 10

parasite membrane, ultimately resulting in greater killing. It is also conceivable that the 11

pair of biocides on the C-terminus of the protein interfere with one another and that a 12

single molecule can be more effective. 13

The concept of using antibody targeting to deliver an antimicrobial payload has been 14

explored for other organisms. Baral et al (4) demonstrated efficacy in nanobody fusion 15

targeting of ApoL to trypanosoma and Fisher et al (18) have shown efficacy in using 16

antibody fusions targeting Fusarium spp. Our results provide further evidence that the 17

ability to build and express recombinant protein molecules which combine elements from 18

the innate and adaptive immune responses offers a novel approach to combinatorial 19

design of antimicrobials. For the first time we have shown an orally delivered 20

recombinant protein immunotherapeutic that is effective at reducing Cryptosporidium 21

parvum infection. Subsequent studies will examine whether this approach is also 22


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


20

effective in a treatment model and if it can bring about reduction in clinical disease in 1

hosts naturally infected by C. parvum. 2

3

4

5

6

7

8

9

Acknowledgements 10

The study was supported by the National Institutes of Health SBIR Grant R44AI56944. . 11


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


21

Reference List 1

2

1. Aley, S. B., M. Zimmerman, M. Hetsko, M. E. Selsted, and F. D. Gillin. 1994. 3

Killing of Giardia lamblia by cryptdins and cationic neutrophil peptides. 4

Infect.Immun. 62:5397-5403. 5

2. Arrowood, M. J. and K. Donaldson. 1996. Improved purification methods for 6

calf-derived Cryptosporidium parvum oocysts using discontinuous sucrose and 7

cesium chloride gradients. J Eukaryot.Microbiol. 43:89S. 8

3. Arrowood, M. J., J. M. Jaynes, and M. C. Healey. 1991. In vitro activities of 9

lytic peptides against the sporozoites of Cryptosporidium parvum. 10

Antimicrob.Agents Chemother. 35:224-227. 11

4. Baral, T. N., S. Magez, B. Stijlemans, K. Conrath, B. Vanhollebeke, E. Pays, S. 12

Muyldermans, and B. P. De. 2006. Experimental therapy of African 13

trypanosomiasis with a nanobody-conjugated human trypanolytic factor. Nat.Med. 14

12:580-584. 15

5. Bowdish, D. M., D. J. Davidson, Y. E. Lau, K. Lee, M. G. Scott, and R. E. 16

Hancock. 2005. Impact of LL-37 on anti-infective immunity. J.Leukoc.Biol. 17

77:451-459. doi:jlb.0704380 [pii];10.1189/jlb.0704380 [doi]. 18

6. Cama, V. A., J. M. Ross, S. Crawford, V. Kawai, R. Chavez-Valdez, D. Vargas, 19

A. Vivar, E. Ticona, M. Navincopa, J. Williamson, Y. Ortega, R. H. Gilman, C. 20

Bern, and L. Xiao. 2007. Differences in clinical manifestations among 21


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


22

Cryptosporidium species and subtypes in HIV-infected persons. J Infect.Dis. 1

196:684-691. 2

7. Carryn S., Schaefer, D. A., Imboden M., Homan, E. J., Bremel, R. D., and 3

Riggs, M. W. Phospholipases and Cationic Peptides Neutralize Cryptosporidium 4

parvum Sporozoite Infectivity by Either Parasiticidal or Non-parasiticidal 5

Mechanisms. International Journal of Antimicrobial Agents 24, 117. 2004. Sixth 6

European Congress on Chemotherapy and Infection and the 24th Interdisciplinary 7

Meeting on Anti-infection Chemotherapy, Paris. 8

8. deGraaf, D. C., E. Vanopdenbosch, L. M. Ortega-Mora, H. Abbassi, and J. E. 9

Peeters. 1999. A review of the importance of cryptosporidiosis in farm animals. 10

Int.J.Parasitol. 29:1269-1287. 11

9. Dillingham, R., A. Lima, and R. Guerrant. 2002. Cryptosporidiosis: 12

epidemiology and impact. Microbes Infect 4:1059. 13

10. Fayer R. 2008. General Biology, In: R. Fayer and L. Xiao (eds.), Cryptosporidium 14

and Cryptosporidiosis. 2nd ed. CRC, Boca Raton. 15

11. Giacometti, A., O. Cirioni, M. S. Del Prete, B. Skerlavaj, R. Circo, M. Zanetti, 16

and G. Scalise. 2003. In vitro effect on Cryptosporidium parvum of short-term 17

exposure to cathelicidin peptides. J Antimicrob.Chemother. 51:843-847. 18

12. Heine, J., J. F. Pohlenz, H. W. Moon, and G. N. Woode. 1984. Enteric lesions 19

and diarrhea in gnotobiotic calves monoinfected with Cryptosporidium species. J 20

Infect Dis 150:768-775. 21


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


23

13. Hong, D. K., C. J. Wong, and K. Gutierrez. 2007. Severe cryptosporidiosis in a 1

seven-year-old renal transplant recipient: case report and review of the literature. 2

Pediatr.Transplant. 11:94-100. 3

14. Huang, D. B., C. Chappell, and P. C. Okhuysen. 2004. Cryptosporidiosis in 4

children. Semin.Pediatr.Infect.Dis. 15:253-259. doi:S104518700400055X [pii]. 5

15. Johansson, J., G. H. Gudmundsson, M. E. Rottenberg, K. D. Berndt, and B. 6

Agerberth. 1998. Conformation-dependent antibacterial activity of the naturally 7

occurring human peptide LL-37. J Biol.Chem. 273:3718-3724. 8

16. McGwire, B. S., C. L. Olson, B. F. Tack, and D. M. Engman. 2003. Killing of 9

African trypanosomes by antimicrobial peptides. J Infect.Dis. 188:146-152. 10

17. Okhuysen, P. C. and A. C. White, Jr. 1999. Parasitic infections of the intestines. 11

Curr.Opin.Infect.Dis. 12:467-472. doi:00001432-199910000-00009 [pii]. 12

18. Peschen, D., H. P. Li, R. Fischer, F. Kreuzaler, and Y. C. Liao. 2004. Fusion 13

proteins comprising a Fusarium-specific antibody linked to antifungal peptides 14

protect plants against a fungal pathogen. Nat.Biotechnol. 22:732-738. 15

19. Riggs, M. W, Schaefer, D. A., and Carryn, S. Control of Bovine Cyptosporidiosis 16

Using Antibody-Based Strategies to Disrupt Parasite Ligand Function and Target 17

Delivery of Parasiticidal Agents. 2004. Chicago, Il. Annual Meeting of the 18

Conference of Research Workers on Animal Disease. 19

20


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


24

20. Riggs, M. W., V. A. Cama, H. L. Leary, Jr., and C. R. Sterling. 1994. Bovine 1

antibody against Cryptosporidium parvum elicits a circumsporozoite precipitate-2

like reaction and has immunotherapeutic effect against persistent cryptosporidiosis 3

in SCID mice. Infect Immun 62:1927-1939. 4

21. Riggs, M. W., T. C. McGuire, P. H. Mason, and L. E. Perryman. 1989. 5

Neutralization-sensitive epitopes are exposed on the surface of infectious 6

Cryptosporidium parvum sporozoites. J Immunol. 143:1340-1345. 7

22. Riggs, M. W., M. R. McNeil, L. E. Perryman, A. L. Stone, M. S. Scherman, and 8

R. M. O'Connor. 1999. Cryptosporidium parvum sporozoite pellicle antigen 9

recognized by a neutralizing monoclonal antibody is a beta-mannosylated 10

glycolipid. Infect.Immun. 67:1317-1322. 11

23. Riggs, M. W. and L. E. Perryman. 1987. Infectivity and neutralization of 12

Cryptosporidium parvum sporozoites. Infect Immun 55:2081-2087. 13

24. Riggs, M. W., A. L. Stone, P. A. Yount, R. C. Langer, M. J. Arrowood, and D. 14

L. Bentley. 1997. Protective monoclonal antibody defines a circumsporozoite-like 15

glycoprotein exoantigen of Cryptosporidium parvum sporozoites and merozoites. 16

J.Immunol. 158:1787-1795. 17

25. Schaefer, D. A., B. A. Auerbach-Dixon, and M. W. Riggs. 2000. Characterization 18

and formulation of multiple epitope-specific neutralizing monoclonal antibodies for 19

passive immunization against cryptosporidiosis. Infect Immun 68:2608-2616. 20


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


25

26. Schnyder, M., L. Kohler, A. Hemphill, and P. Deplazes. 2008. Prophylactic and 1

therapeutic efficacy of nitazoxanide against Cryptosporidium parvum in 2

experimentally challenged neonatal calves. Vet.Parasitol. 3

27. Sorensen, O., K. Arnljots, J. B. Cowland, D. F. Bainton, and N. Borregaard. 4

1997. The human antibacterial cathelicidin, hCAP-18, is synthesized in myelocytes 5

and metamyelocytes and localized to specific granules in neutrophils. Blood 6

90:2796-2803. 7

28. Stockdale, H. D., J. A. Spencer, and B. L. Blagburn. 2008. Prophylaxis and 8

Chemotherapy, In: R. Fayer and L. Xiao (eds.), Cryptosporidium and 9

Cryptosporidiosis. 2nd ed. CRC, Boca Raton. 10

29. Sulzyc-Bielicka, V., W. Kuzna-Grygiel, L. Kolodziejczyk, D. Bielicki, J. 11

Kladny, M. Stepien-Korzonek, and B. Telatynska-Smieszek. 2007. 12

Cryptosporidiosis in patients with colorectal cancer. J Parasitol. 93:722-724. 13

30. Tanaka, T., Y. Omata, A. Saito, K. Shimazaki, K. Yamauchi, M. Takase, K. 14

Kawase, K. Igarashi, and N. Suzuki. 1995. Toxoplasma gondii: parasiticidal 15

effects of bovine lactoferricin against parasites. Exp.Parasitol. 81:614-617. 16

31. Tarver, A. P., D. P. Clark, G. Diamond, J. P. Russell, H. Erdjument-Bromage, 17

P. Tempst, K. S. Cohen, D. E. Jones, R. W. Sweeney, M. Wines, S. Hwang, and 18

C. L. Bevins. 1998. Enteric beta-defensin: molecular cloning and characterization 19

of a gene with inducible intestinal epithelial cell expression associated with 20

Cryptosporidium parvum infection. Infect.Immun. 66:1045-1056. 21


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


26

32. Tzipori, S. and G. Widmer. 2008. A hundred-year retrospective on 1

cryptosporidiosis. Trends Parasitol. 24:184-189. 2

33. Wiersma, E. J. and M. J. Shulman. 1995. Assembly of IgM. Role of disulfide 3

bonding and noncovalent interactions. J.Immunol. 154:5265-5272. 4

34. Xiao, L. and U. M. Ryan. 2004. Cryptosporidiosis: an update in molecular 5

epidemiology. Curr.Opin.Infect.Dis. 17:483-490. 6

35. Zaalouk, T. K., M. Bajaj-Elliott, J. T. George, and V. McDonald. 2004. 7

Differential regulation of beta-defensin gene expression during Cryptosporidium 8

parvum infection. Infect.Immun. 72:2772-2779. 9

36. Zhu, G. 2008. Biochemistry, In: R. Fayer and L. Xiao (eds.), Cryptosporidium and 10

Cryptosporidiosis. 2nd ed. CRC, Boca Raton. 11

37. Ziegler, H. K. and C. A. Orlin. 1984. Analysis of Listeria monocytogenes antigens 12

with monoclonal antibodies. Clin.Invest Med. 7:239-242. 13

38. Zufferey, R., J. E. Donello, D. Trono, and T. J. Hope. 1999. Woodchuck 14

hepatitis virus posttranscriptional regulatory element enhances expression of 15

transgenes delivered by retroviral vectors. J Virol. 73:2886-2892. 16

17

18


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


27

Figure Legends 1

2

3

Table 1. Characteristics of monoclonal antibodies and derived recombinant fusion 4

proteins specific for three different epitopes on C. parvum. All light chains are kappa 5

isotype. N/A indicates the monoclonal antibody used in its native hybridoma-derived 6

form as a control. 7


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


28

Figure 1. Genetic constructs for making antibody fusions using the MLV-based retroviral 1

vector. A. Retroviral construct used to encode antibody heavy chain fused to an 2

antimicrobial. B. Retroviral construct used to encode antibody light chain. LTR=long 3

terminal repeat, EPR=extended packaging region, sCMV=simian cytomegalovirus 4

promoter, SP=signal peptide, VH=Heavy chain variable region, VL=Light chain variable 5

region, CH=Heavy chain constant region, CL=Light chain constant region, RESE=RNA 6

export and stabilization element, (G4S)3=glycine-serine linker, Bioc=biocide 7

8

9

10


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


29

Figure 2. In vitro killing of C. parvum sporozoites by fusion proteins, monoclonal 1

antibodies and combinations of antibody and biocide. Sporozoites were excysted and 2

tested for viability (>98%) then exposed to different compounds for 30 min at 37°C in 3

PBS buffer. Sporozoite viability or death was determined by fluorescein diacetate and 4

propidium iodide staining respectively and data collected by epifluorescence microscopy. 5

A, each component was used at 50 µg/ml except 3E2-G1-LL37 (1.5 µg/ml). B, each 6

component was used at 25 µg/ml, PLA2 and LL37 were used at equimolar 7

concentrations. CHO SN=spent CHO cell medium, Untreated=untreated sporozoites in 8

PBS. MAb indicates use of native hybridoma-derived antibody as control. Means ± SEM 9

and ANOVA of triplicate wells are shown. Bars not connected by the same letter are 10

significantly different (alpha = 0.05). 11


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


30

Figure 3. Fluorescence photomicrographs showing the effect of various fusion proteins 1

and monoclonal antibody controls. A, representative picture of C. parvum sporozoites 2

after an exposure of 30 min to either PBS, CHO cell supernatant, 4H9-G1, 4H9-G2b, 3

18,44 MAb, 4H9-G1-PLA2, 3E2-G1, or 3E2-MAb. B, representative picture of C. 4

parvum sporozoites after an exposure of 30 min to either 4H9-G2b-LL37, 4H9-G1-LL37, 5

3E2-G1-LL37, 3E2-Mhalf-LL37 or 3E2-Mmono-LL37. C, C. parvum sporozoites after 6

an exposure of 30 min to 18.44-G1-PLA2. D, heat-killed sporozoites. Viability or cell 7

death was determined by the addition of fluorescein diacetate (green=alive) and 8

propidium iodide (red=dead) respectively. Each component was used at 25 µg/ml. 9


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


31

Figure 4: In vitro killing of C. parvum sporozoites with low pH-treated 4H9-G1-LL37. 1

4H9-G1-LL37 fusion protein was exposed to various pH conditions for 90 min at 37°C 2

then neutralized to pH 7. Sporozoites were then exposed to the fusions for 30 min at 3

37°C, followed by viability staining and microscopic analysis. Each bar represents a 4

separate pH treatment for 4H9-G1-LL37. Neutral pH-treated fusion protein 166-G2b-5

LL37 and CHO cell supernatant were used as controls. Means ± SEM. 6


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


32

Figure 5: Dose dependent efficacy of different fusion proteins given orally against C. 1

parvum infection in neonatal mice. Groups of 10 neonatal mice were challenged with C. 2

parvum and treated twice a day with fusion proteins for 5 days at either 1.5, 3.8 or 7.7 3

mg/kg/d. Intracellular C. parvum stages in the gut epithelium were quantified by 4

microscopic analysis of gut longitudinal sections. As a positive control hybridoma-5

derived 3E2 MAb was included at 462 mg/kg/d. Mice treated with CHO cell culture 6

supernatant were considered 100% infected. Means ± SEM. 7


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


33

Table 2. Efficacy of fusion proteins compared to their components against infection in 1

neonatal mice. Mice were inoculated with 5x104 purified oocysts by gastric intubation 2

and treated orally with recombinant and control products at 3 and every 12 h for a total of 3

9 treatments. The dose of the biocides given in combination with antibody was adjusted 4

to be equimolar to the antibody. Treatment doses are given in mg/kg/d. Variations in 5

treatment doses among different molecules are of no relevance and were related to 6

product availability. Standard Error uses a pooled estimate of error variance. Treatments 7

not connected by the same letter are significantly different (alpha = 0.05). 8


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


34

Table 3. Testing of 3E2 IgM variants as single or combinatorial treatments in the 1

neonatal mouse model. Mice were inoculated with 5x104 purified oocysts and treated 2

orally with the recombinant monoclonal antibodies or antibody fusions at the doses 3

indicated. For combinatorial treatments each fusion protein was used at half the dose. 4

Standard Error uses a pooled estimate of error variance. Treatments not connected by the 5

same letter are significantly different (alpha = 0.05). 6

7

8

9


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/

Table 1

Antibody Epitope SpecificityHybridoma

IsotypeRecombinant Isotype Peptide Enzyme

mol wt

[kDa]

IgG1 LL37 158

IgG1 sPLA2 IIa 177

IgG1 -- -- 147

IgG2b LL37 160

IgM Monomer LL37 190

IgM Halfmer LL37 95

IgG1 -- -- 147

IgG1 LL37 158

N/A -- -- 970

IgG1 LL37 158

IgG1 sPLA2 IIa 177

N/A -- -- 147

IgG2b LL37 159

IgG2b sPLA2 IIa 178

IgG2b -- -- 148

IgG1

IgG3

IgG2b

IgM

4H9

18.44

166

C. parvum sporozoites

GP25-500

C. parvum sporozoites

CPS-500

L. monocytogenes

cell wall

3E2C. parvum sporozoites

CSL


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/

VHCH

VLCL RESE LTR

RESE LTRBioc X(G4S)3SP

SP

LTR/EPR/sCMV

LTR/EPR/sCMV

A

B

VHCH

VLCL RESE LTR

RESE LTRBioc X(G4S)3SP

SP

LTR/EPR/sCMV

LTR/EPR/sCMV

A

B

Fig 1


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/

Fig 2

B

A

D

C

C

B,C

B,C

B

A

A

A

0 10 20 30 40 50 60

CHO SN

Untreated

4H9-MAb

4H9-G2b-LL37

4H9-G1-LL37

4H9-G1-LL37 aged

3E2-Mmono-LL37

3E2-Mhalf-LL37

3E2-G1-LL37

3E2-G1-SA

3E2 MAb

% KillingA

E

B

C

C

C

D

A

A

A

0 10 20 30 40 50 60

CHO SN

166-G2b-LL37

166-G2b-PLA2

4H9-G1-LL37

18.44 MAb

18.44-G1 + LL37

18.44-G1 + PLA2

18.44-G1-LL37

18.44-G1-PLA2

% KillingB on April 21, 2018 by guest

http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/

Fig 3

Pla

ceho

lder

NO

T for p

rint

Pictu

re s

ubm

itted

as

sepa

rate

TIF

F file


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/

Fig 4

0 10 20 30 40 50

4H9-G1-LL37 pH 2

4H9-G1-LL37 pH 3

4H9-G1-LL37 pH 4

4H9-G1-LL37 pH 5

4H9-G1-LL37 pH 7

166-G2b-LL37 pH 7

CHO-SN pH 7

Killing (%)


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/

0%

10%

20%

30%

40%

50%

60%

70%

0 2 4 6 8

mg/kg/d

Re

du

cti

on

of

Infe

cti

on

[%

]

462

4H9G1-LL37

4H9G2b-LL37

18.44 G1-PLA2

3E2 MAb

Fig 5


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/

Table 2

MAb/Fusion Biocide

CHO supernant negative control NA NA 10 6.40 0.34 0% A,B

4H9-G1-LL37 9.7 9 1.67 0.36 74% D

4H9-G2b-LL37 8.0 9 1.22 0.36 81% D

4H9-G1-PLA2 13.5 9 1.67 0.36 74% D

4H9-G1 + LL37 9.7 0.2 10 4.90 0.34 23% B,C

4H9-G1 + rPLA2 13.5 1.0 9 4.44 0.36 31% C

3E2 MAb 46.5 9 5.44 0.36 15% A,B,C

3E2 MAb 465.0 10 1.50 0.34 77% D

¹Treatments not connected by same letter are significantly different (alpha = 0.05)

Std

Error

% Infection

Reduction ANOVA¹Treatment

Dose [mg/kg/d]

N

Mean

Infection

Score


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/

TreatmentDose

[mg/kd/d]

Mean Infection

Score

Std

Error

Infection

Reduction

CHO SN NA 8.65 0.28 0% A

3E2-G1 7.7 5.4 0.28 38% B

3E2-Mmono-LL37 7.7 4.5 0.28 48% B C D

4H9-G1-LL37 7.7 3.7 0.28 57% D E

4H9-G1-LL37+3E2-Mmono-LL37 3.8+3.8 2.9 0.28 66% E F

3E2-Mhalf-LL37 2.5 1.6 0.28 82% F

ANOVA¹

¹Treatments not connected by same letter are significantly different (alpha = 0.05)

Table 3


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/


http://aac.asm.org/

Dow

nloaded from

http://aac.asm.org/

aac accepts, published online ahead of print on 19 january...

Documents