Interactions Between Human Plasma Components and A Xenogenic Adenovirus Vector: Reduced Immunogenicity During Gene Transfer

original article

© The American Society of Gene Therapy

Interactions Between Human Plasma Components and A Xenogenic Adenovirus Vector: Reduced Immunogenicity During Gene Transfer Matthieu Perreau1,2, Marie-Christine Guérin2, Christian Drouet3* and Eric J Kremer1,2 Institut de Génétique Moléculaire de Montpellier, Montpellier, France; 2CNRS—Universities of Montpellier I and II, Montpellier, France; 3Laboratoire d’Immunologie, Université Joseph Fourier Grenoble 1 and CHU Grenoble, Grenoble, France 1

By the time we are adolescents most of us have been in contact with several of the >50 human adenovirus (HAd) serotypes. These common subclinical infections lead to an efficient anti-adenovirus cross-reacting adaptive immunity. During gene therapy, the ubiquitous anti-­adenovirus humoral response and complement activation will modify and dictate vector biodistribution, as well as the response to the virion and transgene(s). In this study, we assayed the interactions of a xenogenic adenovirus derived from canine serotype 2 (CAV-2) with naturally occurring human antibodies (Abs) and the complement system. In our cohort, we found class G immunoglobulins (Igs) that recognized the intact CAV2 virion and the external virion proteins. However, the majority of donors had low or no neutralizing Abs, class A, or class M Igs. Purified anti-HAd serotype 5 Abs also recognized CAV-2 virion proteins. In addition, in spite of the presence of anti-CAV-2 IgGs, CAV-2 poorly activated the classical and alternative complement cascades. This atypical response was due to a block upstream of the component 3 (C3) convertase and interplay between the component 1 (C1) inhibitor, the C1q–C1r2–C1s2 complex and CAV-2. Our data demonstrate that some xenogenic adenovirus vectors, like CAV-2, could lead to notably different outcomes following systemic delivery. Received 3 April 2007; accepted 23 July 2007; published online 21 August 2007. doi:10.1038/sj.mt.6300289

Introduction Most populations, regardless of location or quality of healthcare, will come into contact with several of the >50 human adenovirus (HAd) serotypes. The most frequent HAd infections in children and adults are due to the serotypes 1, 2, and 5 (HAd subgroup C) encompassing approximately 50% of all isolations.1,2 HAd disease, which can include diarrhea, conjunctivitis, encephalitis, or flu-like respiratory symptoms, is usually self-limiting in a healthy host. However, in newborns and immunocompromised hosts HAd disease can be serious or even fatal.3 Not surprisingly, HAd disease has also become an increasingly prevalent complication

following bone marrow and solid organ transplants due to current immunosuppression regimes.4 Two of the normal outcomes of HAd infection are the generation of a long-lived cellular immunity, which is the primary watchdog of HAd morbidity, and anti-HAd antibodies (Abs). Abmediated immunity includes the generation of neutralizing anitbodies (NAbs) and non-neutralizing opsonizing Abs. Anti-HAd Abs have been studied for decades and the advent of adenovirus vectors rekindled an interest in serotype-specific NAbs. Only more recently have other non-cellular serum components, including non-neutralizing opsonizing Abs, coagulation factors, and the complement pathways, been appreciated for their roles in tropism, efficacy, safety, and duration of HAd-based gene transfer.5 The natural biological role of virus-immunoglobulin (Ig) aggregates (immune complexes or ICs) is a complex one. In the unusual case where a systemic injection of a substantial dose of viral vectors occurs, novel immune-mediated scenarios can be triggered: in particular, rapid and massive accumulation of ­ vector-Ig aggregates. Viral vector-Ig formation will have numerous biological consequences that are rarely advantageous for long-term therapy. For example, ICs can promote vector phagocytosis via the Fc receptor on numerous cell types, allow Ab-dependent cellular cytotoxicity mediated by natural killer cells, or activate macrophages or professional antigen presenting cells, such as dendritic cells.6–8 Notably, maturation of dendritic cells significantly increases the naïve and memory immune response to infected cells.9 ICs can also activate the classical complement pathway either locally or, in the worst-case scenario, in the circulation. There are four known complement pathways: classical (predominantly Ig-mediated), alternative (direct interaction of the complement proteins with the pathogen), mannose-binding lectin, and a novel component 3 (C3)-independent mechanism (also Ig-dependent).10 Typical complement-induced responses are self-limiting, but aberrant global amplification of these responses can contribute to disseminated intravascular coagulation, sepsis, and adult respiratory distress syndrome.11 Complement activation via opsonizing Abs may have been involved in the fatal onset of multiple organ failure of a patient injected with a HAd5 vector.12 To circumvent the interaction with memory immunity, numerous approaches, including xenogenic viral vectors, are

*Present address: National Centre for Angioedema, CHU Grenoble and Université Joseph Fourier, TIMC-IMAG, CNRS UMR 5525, 38043 Grenoble, France. Correspondence: Eric J. Kremer, IGMM CNRS 5535, 1919 Route de Mende, 34293 Montpellier, France. E-mail: [email protected]

1998

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Results Interaction with humoral immunity All donors harbored anti-CAV-2 IgG. Two complementary titration assays, the enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR), were used to identify and quantify anti-CAV-2 class G, A and M Igs within a cohort (n > 50) of blood bank donors. Using a standard ELISA, we found that all members of the cohort harbored IgG that recognized intact HAd5 virions (Supplementary Table S1), while the majority (>90%) also harbored anti-HAd5 IgA and IgM. The distribution and average anti-HAd5 Ig titers (Figure 1a) are consistent with numerous other reports. Similar to the results from Abad et al.23 our quantitative SPR data gave similar interdonor profiles for anti-HAd5 Igs (Figure 1b). Unexpectedly, we found that all donors also harbored antiCAV-2 IgGs. The average anti-CAV-2 IgG titer was approximately twofold lower than that found for HAd5 (Figure 1a). However, the intradonor difference varied from eightfold less (n = 2) to twofold more (n = 3) for anti-CAV-2 as compared to anti-HAd5 IgG. We found identical IgG titers in 16/50, and twofold lower anti-CAV-2 IgG titers in 20/50 (Supplementary Table S1). We Molecular Therapy vol. 15 no. 11 nov. 2007

a 106

Titer

104 103

4 9 11 14 7 4 1

4 10 16 10 5 3 2

14

**

** 1 7 19 19

3 17 16 12

1

ELISA SPR

10

9

102

250 200

8

150

6

100

4

10

50

2 4

1 HAd5 CAV-2

c 400

49

2

41 12

HAd5 CAV-2 HAd5 CAV-2

IgG

IgA

4

15 24 Serum

IgM

d

2

36

400

Serum 36

300

CAV-2

200

Anti-CAV-2 Abs affinity

300

HAd5 RU

RU

300

12

Titer (×104)

105

b

*

RU

being developed. This is being done with the hope that the xenogenic Ads will helpretain some of the advantages of the HAd vector, while ­ bypassing some of its clinical disadvantages. Several years ago we began developing vectors derived from canine serotype 2 (CAV-2),13–16 one of the >60 non-human Ads known. We have earlier shown that in the central nervous system of several species, including the human (ex vivo), CAV-2 vectors preferentially transduce neurons and have an efficient level of axoplasmic transport.16 E1-deleted (∆E1) CAV-2 vectors were less immunogenic than HAd5 vectors in the immunologically naïve rat central nervous system15 and mouse lung.17 Helper-dependent CAV-2 vectors also lead to >1 year in vivo transgene expression in immunocompetent rat central nervous system15 and murine respiratory tract17 without immunosuppression. In addition to the role CAV-2 vectors can play in addressing fundamental ­ neurobiological questions,18–20 our data suggests that helper-dependent CAV-2 vectors could be used for the treatment of some neurodegenerative disorders. However, one can cause more immediate harm via acute vector-induced inflammation than by the normal progression of most disorders. We, and others, have predicted that a memory T-cell response against virion proteins (which is poorly blunted by immunosuppressive drugs21), in combination with complement activation and a relatively strong innate immune response,22 may lead to deleterious side effects in certain tissues. Although adenovirus infections cause relatively species-specific disease, a possible non-neutralizing anti-CAV-2 Ab-mediated immunity due to cross-reactions with other pathogens is an obvious possibility. In addition, the hyper-activation of an innate response by the complement pathway must be explored more completely. In this study, we have examined the interaction of CAV-2 virions with human Abs and the complement system. Our data shows that CAV-2 vectors harbor characteristics distinct from the prototype HAd5 platform. These characteristics may notably modify the response against the transgene and virion in some clinical settings.

Plasma Component Interaction With CAV-2

200 24

100

100

1

2

3

Seconds (×100)

4

5

15

1

2

3

4

5

Seconds (×100)

Figure 1  Anti-HAd5 and CAV-2 immunoglobulin (Ig) titers and affinity. (a) IgG, IgA, and IgM were monitored using classical enzyme-linked immunosorbent assay (ELISA) from 50 donors. The titers of HAd5 or CAV-2 antibodies (Abs) for each class were defined as the reverse of the highest dilution of each serum sample that gives an optical density ≥ negative control average +2 SDs. Bold horizontal bars indicate the average titer. To the left of each symbol is the number of donors with the respective titer. *P < 0.05, **P = 0.004. (b) Comparison of ELISA and surface plasmon resonance (SPR) for inter- and intradonor total serum titers for six donors harboring various titers (as quantified by ELISA) of antiHAd5 and CAV-2 IgG (see Supplementary Table S1). (c) Comparison of the association and dissociation curves of serum #36 to CAV-2 and HAd5 by SPR: NAb titers were 128 (the highest detected) and 1,024 against CAV-2 and HAd5, respectively. (d) Comparison of the association and dissociation curves by SPR of serum #24, which contains the highest anti-HAd5 neutralizing Abs (titer, and of serum #15, which contains an approximately twofold variation in the Ig titers to CAV-2. These sera showed no binding to bovine serum albium (data not shown), and similar to the assay used by Abad et al.23 the virions remained intact when attached to the sensor chip (data not shown). CAV-2, canine adenovirus serotype 2; HAd5, human adenovirus serotype 5; RU, resonance unit.

found a positive correlation between anti-CAV-2 and anti-HAd5 IgG titers (P > 0.001). Using our conditions, the anti-CAV-2 IgAs appeared rarely (1/50), while the anti-CAV-2 IgMs were more frequently found (9/50) (Figure 1a and Supplementary Table S1). However, when present, the anti-CAV-2 IgA and IgM titers were near the minimum level of detection. In this cohort, we found no correlation (R = 0.5) between anti-CAV-2 or anti-HAd5 IgG titers and age (Supplementary Table S1). We previously assayed the CAV-2-induced human memory T-cell response24 and found that the CAV-2 responders had a tenfold lower level of activation compared to HAd5. We did not detect reactive CD8+ cells in any of the donors and less than half of the cohort (42%) harbored proliferating CD4+ cells directed against the CAV-2 virion proteins (versus >85% against HAd5). We therefore compared the humoral response with our memory T-cell data. We found no significant correlation (R = 0.23, n = 8) between the anti-HAd5 or anti-CAV-2 class G, M, or A Ig titers and CAV-2- or HAd5-induced memory T-cell proliferation (data not shown). 1999

© The American Society of Gene Therapy

Plasma Component Interaction With CAV-2

Anti-CAV-2 NAbs are rare and low titers. We had earlier r­ eported that 98% of a cohort from north-central France did not harbor CAV-2 NAb. Due to the widespread anti-CAV-2 IgG ­response in this new cohort, we assayed the sera for CAV-2 NAb. Here, we used a 100-fold more sensitive transduction inhibition assay than the one used previously (see Materials and Methods). CAV-2 and HAd5 NAbs were quantified in sera from >80 ­donors (Supplementary Tables S1 and S2), including 32 who were tested for an anti-CAV-2 cellular immunity (Supplementary Table S2).24 Under these conditions, approximately 70% of the donors harbored no detectable CAV-2 NAbs, whereas >75% harbored HAd5 NAbs (Figure 2). When the donors belonged to the group that we considered as “true responders” (NAb titer ≥16), 12/82 harbored CAV-2 NAb. Including those who were ­“ambiguous responders” (NAb titer >2) the titers were 17-fold lower (on ­average) than that obtained for HAd5 (Supplementary Table S3). No significant correlations (R < 0.05; P < 0.5) were found between: (i) NAbs and memory T-cell proliferation for HAd5 or CAV-2; (ii) CAV-2 and HAd5 NAbs (R < 0.07; P < 0.5); (iii) NAbs titers and age (R < 0.05; P < 0.5); or (iv) the anti-Ad Ab affinity and NAbs titers (data not shown). Anti-HAd5 Abs cross-react with CAV-2 virion proteins. To the best of our knowledge, CAV-2 has never been isolated from, or detected in (e.g., by polymerase chain reaction), human fluids or tissues. Furthermore, by sequence comparison none of the >50 currently identified HAd serotypes appear to be recombinations of human and canine serotypes.25 This suggests that a ­humoral response that recognizes the CAV-2 virion is unlikely to be caused by a wild-type CAV-2 infection, but could be due to a cross-reaction with (an)other Ag(s). Because of the common origin,26 structural similarities and genome organization—the most obvious and likely pathogen is a HAd. Furthermore, due to the widespread prevalence of anti-CAV-2 IgG in this cohort, it is probable that the serotype is common to the region and/or the antigenic sites are shared by a subset of serotypes. 2000

10,000

Responder NAb titer

In contrast to ELISA, SPR also allows one to determine the titer as well as the relative affinity between two molecules. Therefore, based on the above results, we compared sera binding to HAd5 and CAV-2 virions. Using sera from donors that had the highest CAV-2 NAbs titers, we found that the anti-CAV2 and anti-HAd5 Abs had modestly different affinities (Figure 1c). We also compared two sera that had a twofold variation in the anti-CAV-2 and anti-HAd5 Ig titers (Figure 1d). As above, the relative affinities were similar. Because serum contains other components that could also bind Ad virions, we purified total serum Igs to determine if our assay was detecting predominantly Ab affinity. We found similar results (R = 0.95) using whole serum or purified Igs, suggesting that our readout was measuring primarily Ab binding (data not shown). In addition, donors who harbored low anti-HAd5 Igs had a considerably lower resonance unit (RU) (up to sevenfold, data not shown), again suggesting that in this assay we were detecting the affinity of the Abs to the intact virions. No significant correlation (R < 0.2) was found between the affinity and anti-HAd5 and CAV-2 class G, A or M Ig titers.

1,000

100

10

1

HAd5

CAV-2

Figure 2  Anti-HAd5 versus CAV-2 neutralizing antibodies (Abs) titers. HAd5 and CAV-2 neutralizing Abs (NAbs) were quantified using the transduction inhibition assay. The titers were determined by the reverse of the highest dilution of each serum sample that gave a 50% reduction of the number of green fluorescent protein-positive cells. The open triangle and open square correspond to the anti-HAd5 and CAV-2 mean titers of the responders. The dotted line corresponds to the level that we considered significant. CAV-2, canine adenovirus serotype 2; HAd5, human adenovirus serotype 5.

To assay whether the humoral immunity against HAd5 could cross-react with CAV-2, we purified anti-HAd5 Abs from a single donor and from a pool of random donors. In the two samples the relative anti-HAd5 NAb, class G and A Ig titers were comparable (± 2-fold) pre- and post-purification. In the individual donor the anti-HAd5 IgM titers were reduced fourfold. In the pool sample, we did not detect anti-HAd5 IgMs post-­purification. Interestingly, while we obtained a twofold reduction in the IgG titers we found a modest 1.5-fold increase in the NAb titer. In the two samples, the relative anti-CAV-2 NAb and IgG titers (including G1, G2 and G3 titer, data not shown) were comparable pre- and post-purification. The IgA titers were fourfold reduced in the individual donor while they remained steady in the pooled donor. Similar to the case with HAd5, the anti-CAV-2 IgM titers were significantly reduced in the purified fraction. In contrast to the increase in NAb titer found with HAd5 in the pooled donor, we found a 1.5-fold reduction in anti-CAV-2 NAbs. Combined, our data suggested that an anti-HAd5 response cross-reacts with CAV-2 virion. Other non-primate adenoviruses are being assayed as possible gene transfer vectors.27 To determine if the cross-reaction to CAV-2 was exceptional, we repeated the ELISA using serum from six donors and included wells coated with porcine (PAV-3) and bovine (BAV-3) adenovirus virions. We found that, similar to the results obtained with CAV-2, all donors harbored IgGs that recognized porcine and bovine virions (Table 1). The average antiBAV-3 titers were modestly higher (approximately 1.5-fold) than the anti-CAV-2 Ig titers. The anti-PAV-3 titers were ­approximately twofold lower than the anti-CAV-2 Ig titers. In addition, the affinity column-purified anti-HAd5 Igs also ­ recognized PAV-3 and www.moleculartherapy.org vol. 15 no. 11 nov. 2007

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Plasma Component Interaction With CAV-2

Table 1 Class G, A, and M Ig titers against HAd5, CAV-2, BAV-3 and PAV-3 HAd5

Donor

CAV-2

BAV-3

PAV-3

IgG

IgA

IgM

IgG

IgA

IgM

IgG

IgA

IgM

IgG

IgA

IgM

2

16,000

4,000

16,000

4,000

2,000

4,000

4,000

1,000

4,000

2,000

2,000

4,000

38

64,000

4,000

8,000

16,000

1,000

4,000

1,6000

1,000

4,000

8,000

1,000

2,000

42

16,000

4,000

4,000

4,000

1,000

4,000

8,000

1,000

4,000

4,000

1,000

4,000

43

64,000

4,000

2,000

16,000

2,000

2,000

32,000

2,000

2,000

4,000

1,000

2,000

49

16,000

2,000

1,000

8,000

500

2,000

16,000

2,000

1,000

8,000

500

500

51

64,000

8,000

1,000

8,000

500

1,000

16,000

500

1,000

2,000

500

1,000

51-α-HAd5*

16,000

2,000

ND

4,000

ND

500

ND

ND

4000

ND

ND

ND

Abbreviations: HAd5, human adenovirus serotype 5; BAV-3, bovine adenovirus serotype 3; CAV-2, canine adenovirus serotype 2; Ig, immunoglobulin, ND, not detected; PAV-3, porcine adenovirus serotype 3. *51-α-HAd5 = anti-HAd5 Igs purified from serum #51.

Molecular Therapy vol. 15 no. 11 nov. 2007

Total sera binding to virions

160

HAd5 CAV-2

b 1,000

Anti-IgG binding to virion-IC

100 RU

120 80

10

40 51 24 14 21 27 33 38 12 41 43 Donor

c

140

1

51 24 14 21 27 33 38 12 41 43 Donor

Serum binding to individual CAV-2 proteins

Hexon Penton Fibre

120

RU

100 80 60 40 20 51

d

36

500

13

24

14

21 27 Donor

33

38

41

43

Anti-Ig binding to individual CAV-2 protein-IC

16

Hexon Penton Fibre

400 RU

Anti-CAV-2 Igs can recognize the fiber, the penton, and the hexon proteins. Naturally occurring anti-HAd5 Abs react against most virion components. However, the reactions against hexon, penton base, and fiber (the major external virion proteins28) are primarily responsible for neutralization and opsonization.29–31 To identify the CAV-2 virion proteins that are recognized by the anti-HAd5 cross-reaction, a combination of western blot and SPR assays was performed using sera from >10 donors (including that which harbored the highest CAV-2 NAbs titers). Initially, we bound HAd5 and CAV-2 to the sensor chip and injected whole serum so as to determine binding (Figure 3a). We found that all donors harbored serum components that bound HAd5 and CAV-2. We then injected anti-IgG Abs (as well as anti-IgA and IgM Abs, data not shown) to determine if the binding was Ig-specific (Figure 3b). We found that in some donors (e.g., #14 and 27) binding was not exclusively due to IgGs (we found no anti-class A or M Ig binding to these two sera). We then equilibrated the sensor chip with an equal RU for each major external virion protein (this corresponds to an approximate ratio of 1 trimeric hexon, 1.16 penton bases, 1.70 trimeric fibers). In this assay, we found serum components recognizing the three major external capsid proteins (Figure 3c). We detected binding of the hexon and fiber in all donors, whereas the penton base was detected in 10/12. Hexon, which is the most highly conserved external adenovirus capsid protein, also consistently gave the highest RU values. Our analysis reflects the response against the native protein and therefore some linear as well as conformational epitopes. In addition, by binding the proteins to the chip, several additional epitopes become exposed that are normally buried in the virions. As expected, this will

a RU

BAV-3 virions (Table 1). Together, this data revealed that 6/6 donors harbored Igs that recognized three xenogenic Ads, and that Abs that recognize one of the most prevalent HAd serotypes (HAd5) also recognize intact CAV-2, PAV-3 and BAV-3 virions. Although it might be possible, we believe that it is unlikely that the six donors from this region had all been in close contact with dogs, pigs, and cattle. We therefore propose that this humoral response was simply a primary response against HAd serotypes that cross-reacted with xenogenic Ads, and was not a specific response to CAV-2, PAV-3 and BAV-3.

300 200 100 G A M G A M G A M G A M G A M G A M G A M G A M G A M G A M G A M G A M

51

36

13

24

14 21 27 33 Donor and Ig class

38

41

43

16

Figure 3  Interdonor levels of anti-CAV-2 external capsid proteins immunoglobulin (Ig) titers. (a) HAd5 and CAV-2 were immobilized on the surface plasmon resonance sensor chip and sera from 10 donors were assayed for binding. The data are the mean of three assays ± SEM. (b) Binding of anti-IgG antibodies to HAd5 and CAV-2-ICs. HAd5 and CAV-2 were immobilized on the sensor chip, serum from ten donors was injected, and then anti-class G antibodies were injected. The data are the mean of two assays. (c) External CAV-2 capsid proteins were fixed to the sensor chip and sera from 12 donors were assayed for serum binding. The data are the mean of three assays ± SEM. (d) Binding of anti-IgG, -IgA, and -IgM antibodies to external CAV-2 protein-IC. The data are the mean of two assays. IC, immune complex; RU, resonance unit CAV-2, canine adenovirus serotype 2; HAd5, human adenovirus serotype 5.

lead to greater Ab binding in some sera (e.g., serum #14 poorly binds CAV-2 virion but readily binds the CAV-2 hexon). Using western blot analysis (denatured proteins), we also found a cross-reacting response against the three major external CAV-2 virion proteins (Supplementary Table S4). As mentioned before, using intact virions,we detected primarily Ab binding by SPR. However, fiber may also be responsible for binding other serum components.32 In addition, the RU values were relatively low for the penton and fiber binding. We therefore extended our analysis to determine if Igs were in fact binding the 2001

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Plasma Component Interaction With CAV-2

2002

Classical pathway

80 60 40 20 1

Alternative pathway 100 80 60 40 20

2.5 5 pp/ml (×1011) HAd5

c

b % vs. control

% vs. control

100

1

CAV-2

Alternative pathway 100

2.5 5 pp/ml (×1011) HAd5

d

CAV-2

Lectin pathway 0.6 0.5

80

0.4

60

A490

Activation of complement CAV-2 poorly activates the classical, alternative, and ­lectin pathways. Although anti-HAd Ab-mediated immunity, and particularly neutralizing activity, has been studied for decades, ­activation of the classical and alternative complement pathways by HAd5 has only recently been reported.12,33,34 Due to the widespread presence of anti-CAV-2 IgGs, we assayed the classical, ­alternative, and lectin pathways. Consistent with previous results,12 we found that HAd5 was a potent complement activator. As little as 1 × 109 of the physical particle (pp)/ml (data not shown) activated the classical (Figure 4a) and 2.5 × 1011 pp/ml activated the alternative pathway (Figure 4b). In contrast, we found that CAV-2 showed no notable activation of either pathway (Figure 4a and b). To accentuate the activation, we also assayed the alternative pathway amplification loop. Under these conditions we found that >70% of the activity was consumed at the lowest dose of HAd5 (Figure 4c). In the presence of antiCAV-2 IgGs (at a titer that was fourfold lower than that of HAd5) and IgMs (at a titer that was identical to that found for HAd5), the highest dose of CAV-2 had no notable activation of the amplification loop. Finally, it was also seen that neither HAd5 nor CAV-2 activated the lectin pathway (Figure 4d). Together, this data revealed that the difference between the complement activation of CAV-2 and HAd5 was approximately 4,000-fold. We extended our analysis to try to understand why the classical pathway was poorly activated when combining CAV-2 and antiCAV-2 Abs (for summary of the relevant titers in these plasma see Supplementary Table S5). Using the above assays, we were unable to discriminate between (i) the lack of the classical pathway activation due to regulations of the system and (ii) a lack of initiation of complement component 1 (C1) activity. The classical pathway (Supplementary Figure S1) usually starts with the activation of the C1 complex “C1qr2s2” by IC. Using a combination of physical and biochemical assays, we quantified the binding of C1q to the “virus and anti-virion Ab complex” and the activation of C1 complex. First, we bound HAd5, CAV-2, or C1q to the SPR sensor chip. We then sequentially passed (i) serum, (ii) serum pre-incubated with HAd5, or (iii) serum pre-incubated with CAV-2 over the chip (Figure 5a). Injection of serum allowed us to generate an IC on the sensor chip. We then injected C1q to measure how well it binds to the chip-bound ICs. We found that C1q efficiently bound immobilized HAd5- and CAV-2-IC (Figure 5b). Second, the soluble ICs, formed when serum was pre-incubated with HAd5 or CAV-2, bound to immobilized C1q (Figure 5a), although to

a

% vs. control

individual proteins (Figure 3d). We found that when probing the plasma-protein complexes, the hexon appeared to be binding Igs, suggesting that the modest RU values found against the fiber and penton base could reflect a low cross-reaction and/or binding of other serum components.5 Together, this data should be viewed keeping in mind that while most of the fiber protein is exposed on the virion, most of the hexon and penton are buried in the virion. The exposed side of the hexon also contains the hypervariable regions, which are responsible, in part, for the serotypespecific neutralization. Interestingly, all donors harboring CAV-2 NAbs showed reactivity against the denatured CAV-2 fiber protein (Supplementary Table S4).

40

0.3 0.2

20

0.1 1

2.5 5 pp/ml (×1011) HAd5

CAV-2

1

2.5 5 pp/ml (×1011)

Mock

HAd5

CAV-2

Figure 4  Activation of the complement pathways by HAd5 or CAV-2 vectors. (a) Activation of the classical pathway was monitored using the ability of human complement to lyse sheep erythrocytes. Pooled plasma samples from 20 donors were incubated for 90 minutes with either HAd5 or CAV-2 and then added to erythrocytes. The kinetics was recorded at 660 nm. Consumption of the complement activity by the viruses provokes a “hemolytic delay”, expressed in percent of complement activity remaining (percentage versus reference plasma). (b) Activation of the alternative pathway was monitored using rabbit erythrocytes in the presence of ethylene glycol tetraacetic acid (EGTA) supplemented with Mg2+ (blocks classical and mannose-binding lectin (MBL) pathways). (c) The amplification loop, as measured by activation of the alternative pathway in the absence of EGTA and Mg2+, was measured by the remaining component 3 (C3) convertase activity. (d) Activation of the lectin pathways was determined using MBL-deficient plasma and ­mannan-coated plates as activator. Results are expressed as A490, representative of bound C4b. Error bars correspond to the average ± SEM. pp/ml = physical particle/ milliliter; CAV-2, canine adenovirus serotype 2; HAd5, human adenovirus serotype 5.

a lower but noteworthy level for CAV-2. In addition, these data also re-confirmed that CAV-2 and HAd5 were competing for a common population of Igs (Table 2). This is demonstrated by the reduction of the RU value when comparing the binding of serum versus serum pre-incubated with HAd5 or CAV-2 to immobilized HAd5 or CAV-2. From these data we concluded that CAV-2-ICs were formed and that C1q could bind, demonstrating that the classic complement pathway block was further downstream. Using a biochemical test, we assayed the catalytic activity of C1 using our reference plasma challenged with various doses of antigens. Under these conditions, CAV-2-IC promoted the activation of C1s (Figure 5c). This data suggested that CAV-2-IC could initiate classical convertase activation and the block was downstream of C1s activity. We then indirectly tested the C3 convertase activity via the production of C3a-desArg (Supplementary Figure S1). In contrast to HAd5-IC, we found that CAV-2-ICs were notably less potent in the induction of C3a-desArg (Figure 5d). This data www.moleculartherapy.org vol. 15 no. 11 nov. 2007

© The American Society of Gene Therapy

Plasma Component Interaction With CAV-2

a 10

b

50

Pre-purification

40 RU (×102)

8 RU (×102)

Table 2  HAd5 and CAV-2 Abs pre- and post-purification

C1q binding to ICs

6 4

D1

HAd5

CAV-2

HAd5

30

NAb

1,024

ND

1,024

ND

20

IgG

25,600

25,600

12,800

12,800

IgA

3,200

3,200

1,600

800

IgM

1,600

1,600

400

400

256

16

360

10

10

2

HAd5 CAV-2 Clq Protein/virus immobilised

NAb

HAd5

6 5 4

CAV-2

µg/ml

2

*

3 2

1

5 × 109

5 × 109

pp/ml

Role of C1INH

100

5 × 1010 1.25 × 1010

e

10

5 × 1010 5 × 108

2.5 5 7.5 pp/ml (×1011)

CT

1 5 × 10

C1s (µg)

3

d

C1s activity

C3a-desArg production * HAd5 CAV-2

8

4

EGTA Mg++

% vs. control

80 60 40 20

rC1INH

−

−

+

Figure 5 C1INH-based block of CAV-2 vector activation of the complement pathway. (a) HAd5, CAV-2, and C1q were fixed to individual channels of a surface plasmon resonance sensor chip and the binding of serum from a single donor was quantified (white bars). The sensor chip was stripped, and we quantified binding of serum pre-incubated with HAd5 (gray bars) or CAV-2 (black bars) to the immobilized samples. (b) HAd5, CAV-2, and C1q were fixed to a sensor chip; serum was passed over the samples to generate immune complexes (ICs), and then soluble C1q was passed over the ICs. Total C1q binding is shown (the resonance units (RUs) from the HAd5, CAV-2 and antibodies have been subtracted). (c) Dose effect of component 1s (C1s) activation by HAd5 versus CAV2: the reference plasma mix was incubated with the indicated doses and C1s activation was monitored as described in Methods. The results are expressed in micrograms of activated C1s. (d) C3a-desArg production following incubation with various doses of HAd5 or CAV-2. Plasma was pre-treated with ethylene glycol tetraacetic acid (EGTA) or EGTA supplemented with MgCl2 and incubated with increasing doses of virions (5 × 1010 physical particles (pp)/ml). The star indicates samples with equal quantities of CAV-2 and HAd5 and the horizontal dotted line is the background level. (e) Role of C1INH in the regulation of CAV-2 IC stimulated classical complement pathway: CAV-2 incubated with plasma from a C1INH+ donor, CAV-2 incubated with plasma from C1INH-­deficient (C1INHdef) patient, or CAV-2 incubated with plasma from C1INHdef patient and supplemented with purified C1INH. CAV-2, canine adeno­ virus serotype 2; HAd5, human adenovirus serotype 5.

suggested that the primary regulation(s) was upstream of the formation of C3 convertase. Under physiological circumstances, the classical complement pathway activation is regulated at several levels. At the C1 stage, the only known circulating regulatory protein is the C1 inhibitor Molecular Therapy vol. 15 no. 11 nov. 2007

CAV-2

MD

HAd5-IC CAV-2-IC

Serum Serum pre-incubated with HAd5 Serum pre-incubated with CAV-2

c

Post-purification

IgG

64,000

16,000

32,000

16,000

IgA

2,000

500

2000

500

IgM

500

500

ND

ND

Abbreviations: CAV-2, canine adenovirus serotype 2; D1 = donor 1; HAd5, human adenovirus serotype 5; Ig, immunoglobulin; NAb = neutralising antibody; ND = not detected; MD = mix of 15 donors.

(C1INH).35 To determine the role played by this component in the control of the C1 complex activation by CAV-2-IC, we assayed the plasma of a C1INH-deficient (C1INHdef ) patient (Figure 5e). In the C1INHdef serum we found that the classical pathway was moderately impaired with 74% activity remaining (data not shown; this value was then used as “100%”). When we incubated the C1INHdef plasma with CAV-2-IC, 40% of the classical complement activity was lost. This data suggested that in the absence of C1INH, CAV-2-IC could induce the classical complement pathway. We then supplemented the C1INHdef plasma with a physiological concentration of purified C1INH. Under these conditions CAV-2-ICs were no longer able to significantly induce complement activation. From this, we concluded that the normal role of C1INH was being modified by the CAV-2-ICs. A novel mechanism would be a direct interaction between C1INH and the CAV-2 virion. However, we could not detect C1INH binding to CAV-2 virion by SPR (data not shown). In addition, co-incubation of CAV-2 and HAd5 virion also led to activation of complement, suggesting that CAV-2 was not an active inhibitor (data not shown). Further analysis will be needed to understand this complex mechanism of complement inhibition by CAV-2.

Discussion In this study, we evaluated some of the immune response parameters that are relevant to the use of xenogenic virions in the clinical setting. We previously assayed the interaction of CAV-2 with the human memory T-cell immunity24 and dendritic cells.36 In contrast to HAd5, we found that a minority of the cohort harbored reactive (proliferation, activation, and TH1 differentiation) T cells against CAV-2. We showed that the CAV-2 virion also poorly transduced or induced the functional maturation of human dendritic cells.36 These earlier results together prompted us to try and determine whether CAV-2 could interact with specific plasma components. In our cohort, we found the widespread presence of antiCAV-2 IgG, while IgM and IgA were rarely found or only found at ­background levels. No, or low, NAb titers have been reported in sera for several xenogenic14,27,37 or rare HAd serotypes.1,38,39 Although we detected anti-CAV-2 NAbs in approximately onethird of the responders, the average anti-CAV-2 NAb titers were 2003

Plasma Component Interaction With CAV-2

significantly lower than that found for HAd5. Surprisingly, we found that the relative affinity of the Ab response against HAd5 and CAV-2 was only modestly different. Sera that harbored neutralizing anti-CAV-2 Abs routinely recognized the denatured CAV-2 fiber, suggesting that neutralization was preventing receptor attachment and not intracellular disassembly. We suggest that the origin of the anti-CAV-2 response is due, in part, to a cross-­reaction to HAd because purified Igs and anti-HAd5 Abs recognized intact canine, porcine, and bovine adenovirus virions. Because complement activation via immune-complex formation poses a risk immediately postinstillation, we assayed its interaction with CAV-2. In contrast to HAd5, we found that CAV-2 did not activate the classic or alternative complement pathways. Our data suggested an inhibition of complement between C1 activation and C3 convertase activity. Using plasma from a C1INHdef patient, we found a role based on C1INH interaction. Pre-existing humoral immunity: The high affinity anti-CAV-2 IgG titers were unexpected. It is not possible to formally exclude the scenario where CAV-2 could have infected humans, although we believe this is improbable. The prolonged relationship between humans and dogs has provided numerous opportunities for cross-species infections.40 In the early 1970s, one proposed link was CAV-1-induced hepatitis in humans. Instead of showing a relationship between CAV-1 infection and hepatitis, Smith et al. showed that 80% of a cohort harbored anti-CAV-1 NAbs.41 Surprisingly, this percentage was not greater in serum taken from veterinary workers,42 showing that closer contact with dogs did not increase the anti-CAV-1 NAbs titer. In addition, the same study showed one-way cross-reaction between CAV-1 and HAd8 NAbs in human sera, implying that CAV-1 readily infects humans. Based on our experience and data, we believe that these early studies should be re-evaluated. Members of this lab who have worked with enormous quantities of CAV-2 for years still do not harbor neutralizing anti-CAV-2 Abs. Furthermore, we believe that since the IgG titers recognize BAV-3 and PAV-3, this supports our conclusion of a cross-reaction and not a primary infection. The cross-reaction could be explained, at least in part, by the level of conserved sequence (e.g., 67, 54, and 13% sequence identity between HAd5 and CAV-2 for the hexon, penton base, and fiber proteins, respectively). The interesting, and possibly predictable, difference in the purification of anti-HAd5 compared to anti-CAV-2 NAb (a 1.5-fold increase versus a 1.5-fold decrease, respectively) is consistent with our understanding of the divergence in the fiber knob sequences that interacts with the coxsackievirus and adenovirus receptor.43 If NAbs are directed against this region in the HAd5 knob, it is probable that they do not recognize the coxsackievirus and adenovirus receptor-interacting domain in the CAV-2 fiber knob. The complement pathways: Opsonizing Abs form an essential link between the innate humoral response, scavenger cells, antigen presenting cells, and adaptive cellular immunity. The three common complement pathways are tightly intertwined in vivo, and converge upon one central component, C3 protein.44 For example, the lectin pathway can initiate the cascade, while the alternative pathway can act as an amplifier for the classical ­pathway. ­Complementgenerated peptides influence several acute-phase inflammatory 2004

© The American Society of Gene Therapy

responses, including leukocyte chemotaxis and degranulation, inflammatory cytokine/chemokine secretion, and coagulation pathway activation.45 Not surprisingly, these same responses have been reported post-HAd5 vector instillation. HAd1 (subgroup C), HAd3 (subgroup B), HAd5 (subgroup C), but not HAd4 (species E) activate the classical pathway.12,33,34 However, anti-HAd4 Abs were not found in the above report. In contrast, CAV-2 did not activate the classical or alternative complement pathways in the presence of anti-CAV-2 Abs. Although under normal conditions the complement system is tightly regulated, massive intravenous vector injections could relieve inhibitions of complement. HAdICs have an intrinsic and “normal” potential to activate complement that over-rides the regulation of two complement pathways. There are a few possible controls between C1 activation and the production of C3a-desArg. One of the most obvious was the role of C1INH, the only known circulating control of C1. Under normal conditions, the classical pathway initiated by C1 activation is aborted, in part due to C1INH. Based on our observations, it is premature, and beyond the scope of this initial study, to speculate how C1INH and CAV-2 interact. Although our initial assay suggested that CAV-2 and C1INH do not interact directly, we cannot exclude this possibility. Other plasma components may be needed to stabilize the complex or modify the virion or C1INH. Clinical relevance: Viral vector ICs, without NAbs, are poorly appreciated, tested or understood. Very few studies have addressed non-neutralizing opsonizing Abs that could promote complement activation (i.e., acute-phase inflammatory responses), vector phagocytosis via the Fc receptor, inflammation and/or immunomodulation via the interaction with antigen presenting cells. Most pre-clinical studies in rodents have not been designed to evaluate this scenario. With the advent of xenogenic13,27 and hybrid46 Ad vectors this scenario will become frequent. The clinical relevance of non-neutralizing opsonizing Abs lies in the fact that cells that express Fc receptors efficiently internalize IC. The Fc receptor family includes high- and lowaffinity receptors for the Fc fragment. The low-affinity receptors (FcγRII and FcγRIII) clear IC from tissue and serum and enhance the immune response to the antigen contained in the IC.47 In the event that viral capsids gain entry via Fc receptors,48 this can lead to particularly strong immune responses to virus-encoded antigens. Leopold et al. showed that cells deficient in coxsackievirus and adenovirus receptor, but positive for the Fc receptor can endocytose Ad–Igs complexes and transgene expression can be detected. Using a recombinant coxsackievirus and adenovirus receptor-Fc hybrid fusion protein incubated with HAd5 virion, Meier et al. found that endocytosis was responsible for uptake of approximately 10% of the virion in epithelial cells.49 Vector–Ig complexes will therefore notably modify the vector biodistribution. This could be particularly dangerous in lymphoid tissues where a high concentration of Fc receptor–bearing cells resides. It is also important to note that these complexes will vary among individuals based on the isotype and the epitopes recognized. The data also suggests a possible use of some xenogenic Ad vectors as Ad-based vaccines: e.g., pre-complexing the vectors with serum containing non-neutralizing opsonizing Abs, prior to injection, should target and express transgenes in antigen presenting cells in vitro and in vivo. www.moleculartherapy.org vol. 15 no. 11 nov. 2007

© The American Society of Gene Therapy

Our data shows that CAV-2 vectors circumvent some of the drawbacks associated with systemic delivery of HAd vectors; this conclusion differs notably from traditional concepts of HAd immunogenicity.

Materials and Methods Vectors, viruses and cell lines. CAVGFP and CAVβgal are ∆E1 CAV-2 vec-

tors and contain an enhanced green fluorescent protein (enhanced GFP) or lacZ expression cassette.14 AdGFP and Adβgal are ∆E1/E3 vectors derived from HAd5 containing the same enhanced GFP or lacZ expression cassette. CAVGFP and AdGFP have a physical particle to infectious unit ratio of 3:1 and 10:1, respectively. CAVβgal and Adβgal have a physical particle/ infectious unit ratio of 10:1 and 15:1, respectively. AdGFP and Adβgal were propagated in 293 or 911 cells. CAVGFP and CAVβgal were propagated in DKCre cells. Cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Auckland, New Zealand) supplemented with 10% (vol/vol) fetal calf serum (Sigma-Aldrich, Saint Quentin, France) and 2 mmol/l glutamine (Merck, France). Porcine serotype 3 (PAV-3) and bovine serotype 3 (BAV-3) were gifts from Bernard Klonjkowski (Ecole Vétérinaire d’Alfort, Maissons-Alfort, France). We purified all vectors by banding on CsCl density gradients. The vectors were endotoxin free. Donor samples. Sera were collected in uncoated tubes from healthy

donors from the Etablissement Français du Sang (Montpellier, France). When necessary, sera were decomplemented at 56 °C for 25 minutes and stored at −20 °C. Anti-HAd5 Abs were isolated using CNBr beads with covalently linked AdGFP using 5 ml of 0.45 µm-filtered plasma from one donor who was seropositive for HAd5, and from a mixed pool of 15 donors. Anti-HAd5 Abs were eluted from the column with glycine buffer (pH 2.2), precipitated in NH4SO4 (0.4 mg/ml), resuspended in 250 µl HEPES (50 mmol/l, pH 7.2), dialyzed against phosphate-buffered saline (PBS) (Invitrogen, Cergy-Pontoise, France) and stored at −80 °C. We used Western blot analysis to determine the efficacy of the anti-HAd5 Abs purification. Briefly, we used the serum, flow-through, wash steps and columnbound fractions as probes for HAd5 proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Greater than 95% of the antiAd5 Abs were removed from the serum (i.e., not found in the flow-through and wash fractions) and attached to the column. The relative concentration of the anti-HAd5 Abs compared to the sera was significantly higher in the column-bound eluted fraction. Total Igs were purified from serum using a commercially available kit (Ademtech; Pessac, France). We have no data concerning the anonymous donors’ previous interaction with dogs, pigs, or cattle. Detection of anti-HAd5 and anti-CAV-2 NAbs. We assayed sera for

the ability to inhibit CAVGFP or AdGFP transduction using enhanced GFP expression as the readout. In our previous assays,14 we used 10 µl of serum and 5 × 107 pp of CAVGFP or AdGFP in a final volume 100 µl of medium for 1 hour at room temperature before incubation with 2 × 105 911 cells/well. In this study, we used DKCre cells to titer CAVGFP, which allowed us to lower the number of input particles a 100-fold to generate a TCID20 for the same volume of serum. Cells (911 or DKCre) were seeded at 105 cells/well in 24-well plates and incubated for 24 hours. Sequential twofold dilutions of sera were pre-incubated with a TCID20 (5 pp/cell) of each vector for 15 minutes at room temperature, and then incubated with 911 or DKCre cells. The plates were then gently agitated for several hours to increase vector–cell interaction. We also performed a standard titration of each virus using 1.25, 2.5, 5, 10, or 20 pp/cell. After 24 hours at 37 °C, cells were washed with PBS, and GFP expression of transduced cell was analyzed on a FACS Calibur (BD Biosciences, San Jose, CA) and data analysis was performed using CellQuest software (BD Biosciences). The reverse of the highest dilution of each serum sample that gave a 50% reduction of the number of GFP-positive cells was reported as the NAb titer. Molecular Therapy vol. 15 no. 11 nov. 2007

Plasma Component Interaction With CAV-2

IgA, IgM, and IgG titers and affinity

ELISA: CAVGFP and AdGFP were diluted to 109 pp/ml in carbonate buffer (1.59 g Na2CO3 and 2.93 g NaHCO3 in 1 l H2O, pH 9.6), and 100 µl of this solution was applied to each well of a 96-well flat bottom plate (Nunc maxisorp, Roskilde, Denmark). The plates were incubated overnight at 4 °C. Subsequent steps were performed at room temperature. The plates were rinsed three times with 0.05% Tween (Sigma-Aldrich, Saint Quentin, France) in PBS, and once with deionized water. Non-specific binding was reduced by the addition of PBS supplemented with 10% (vol/vol) fetal calf serum for 2 hours. The plates were again rinsed as above. Serum samples were initially diluted 200- or 500-fold in PBS and subsequently diluted 2-fold. One hundred microliters of each dilution was added to the plate, incubated for 2 hours and rinsed as above. Anti-Ad IgA, IgM, and IgG (Sigma) were detected using 100 µl of a 10,000-fold dilution of rabbit antihuman Ig conjugated to horseradish peroxidase in PBS for 2 hours. The plates were rinsed again, and 100 µl per well of tetramethylbenzidine (BD Biosciences, San Jose, CA) was added and incubated for 30 minutes. The reaction was quenched by the addition of 50 µl of 0.25 N HCl. Absorbance was measured at 450 nm on an ELISA plate reader. The titer of HAd5 or anti-CAV-2 Abs for each isotype was defined as the reverse of the highest dilution of each sample that gives an optical density ≥ negative control average + 2 SDs. SPR: Isolated hexon, and recombinant penton base and fiber proteins from CAV-2 (ref. 24) (1 µg/ml, in 20 mmol/l sodium acetate, pH 3.5), and HAd5 and CAV-2 virion (5 × 108 pp/ml in 20 mmol/l sodium acetate, pH 3.5) were immobilized onto the surface of a CM3 sensor chip (flow rate of 5 µl/ min) using an amine coupling kit (BIAcore AB, Uppsala, Sweden). A blank reference flow cell, used to correct for refractive index changes and nonspecific binding, was prepared using 100 µg/ml bovine serum albium in the same buffer and the same flow rate as that used to prepare sensor chips immobilized with virus. Sera and secondary Abs (anti-IgG, IgM, IgA) were diluted 1/10 and 1/100 with 10 mmol/l HEPES, 150 mmol/l NaCl, 3.5 mmol/l EDTA, 0.005% P-20 surfactant (BIAcore AB, Uppsala, Sweden), 1 mg/ml carboxymethylated dextran (pH 7.4; Fluka Biochemicals, St. Louis, MO). Running buffer (10 mmol/l HEPES, 150 mmol/l NaCl, 3.5 mmol/l EDTA, 0.005% P-20, pH 7.4) was filtered and degassed before use. Each sample (80 µl) was injected at a flow rate of 20 µl/min and regeneration of the surface between samples was performed with 5 µl of 150 mmol/l guanidine–HCl, 5 mmol/l NaOH. Report points were placed 10 seconds before sample injection (baseline) and 30 seconds post-injection. The net RU was obtained by subtracting the baseline from the sample response. We used a BIAcore 2000 (BIAcore AB, Uppsala, Sweden). Complement assays

Classical, lectin and alternative pathways: Activation of the classical and alternative complement pathways were monitored using current clinical protocols based on the ability of human complement to lyse sheep erythrocytes sensitized with rabbit IgG (classical pathway) or rabbit erythrocytes (alternative pathway) in the presence of magnesium and ethylene glycol tetraacetic acid (EGTA), which blocks classical and mannose-binding lectin pathways. The amplification loop: once the classic pathway is activated, C3b can also be used by the alternative pathway, which in turn consumes factor B. To control for auto-activation due to the dilution of inhibitors, the reference sample was incubated with an equivalent volume of PBS. In addition, all experiments were performed at both 37 and 4 °C. Briefly, pooled citrate plasma samples from 50 donors were mixed and stored at −80 °C (reference sample). The plasma reference sample was pre-incubated for 30 minutes at 37 °C with either HAd5 or CAV-2 at various doses ranging from 5 × 1010 to 5 × 1011 pp/ ml (final volume of 200 µl) in the presence of 5 mmol/l MgCl2, 3 mmol/l EGTA, pH 7.4 (alternative pathway) or in Hanks’ buffer supplemented with 2.5 mmol/l CaCl2, and 1.5 mmol/l MgCl2 (classical pathway). To measure the complement hemolytic activity, 150 µl (alternative pathway) or 25 µl (classical pathway) of each sample were subsequently added to

2005

© The American Society of Gene Therapy

Plasma Component Interaction With CAV-2

1 ml erythrocytes (109 cells/ml). The complement activity, corresponding to the time required for 50% total hemolysis, was recorded by the A660 (reference values: 175 ± 50 seconds and 190 ± 40 seconds, respectively for classical and alternative complement pathways). CsCl (d = 1.34) dialyzed against PBS supplemented with 10% glycerol was used as volume control. The consumption of complement activity by the viruses was expressed in percent of complement activity remaining (percentage versus reference sample), and calculated using the formula: ((time required to obtain 50% of erythrocyte haemolysis for the control (C)) (time required to obtain 50% of erythrocytes hemolysis for the sample (C1))−1[100]. The activity of the lectin pathway was determined in samples (1/10 dilution in the presence of a 1/5 dilution of a mannose-binding lectin-deficient plasma, 5 mmol/l CaCl2 and 1.5 mmol/l MgCl2) from the C4 cleavage by the quantification of C4b deposited onto mannan-coated microplates. Data were expressed as A490 representative of bound C4b. Dose–response effect on C1 activation by HAd5 and CAV-2: A 1/12 dilution of plasma (the dilution able to arrest C1 control) in Hanks’ buffer supplemented as above for classical pathway (total volume 100 µl) was incubated at 37 °C with various amount of vector (in 20 µl final volume) for 30 minutes. An equivalent volume of CsCl dialyzed against PBS supplemented with 10% glycerol was used as negative control, whereas 1.4 µg purified C1s protease (57 nKatal/mg) diluted in 20 µl complete Hanks’ buffer was used as positive control. The reaction starts when activated samples (50 µl) were added to 1 ml substrate mixture (1 mmol/l BAEe-HCl, 0.2 g/l ethanol-DH, 1 mmol/l NAD+, 1 mmol/l glutathione, 225 mmol/l semicarbazide-HCl, 20 mmol/l Tris–HCl, 41 mmol/l glycine at pH 8.5). The kinetics was performed for 15 minutes with a recording of A340; enzymatic activity was calculated taking E340 NADH 6.220 (mol/l)−1 cm−1. C3a production: C3a-desArg levels were monitored as previously described by Cichon et al.12 Briefly, plasma from citrate blood was obtained by centrifugation (5 minutes, 800g, 4 °C) and kept on ice. Plasma samples (350 µl) were mock or pre-treated with EGTA (1/10 volume, 100 mmol/l EGTA), or EGTA supplemented with 3 mmol/l MgCl2 prior exposure to Ads, and viral suspensions were added to plasma samples and incubated for 90 minutes at 37 °C. C3a production was stopped by the addition of EDTA (1/10 volume 100 mmol/l EDTA). Levels of C3a-desArg were determined using a commercial ELISA (BD Biosciences, San Jose, CA). C1q and C1 inhibitor (C1INH): The activation of a plasma sample from a C1INHdef patient (C1INHdef ) genetically identified as a type I hereditary angioedema was compared to a control plasma sample. The C1INHdef patient contained a subnormal level of C4 protein and normal hemolytic activity, and 50 µg/ml C1INH (18% reference value). The samples were incubated for 90 minutes with 1 × 1011 pp of CAV-2 (or HAd5, data not shown) and activity of the classical pathway was assayed as above. The pathological sample was assayed after supplementation with purified C1INH (final concentration 300 µg/ml).

Acknowledgments We acknowledge the exceptional aid of Christine Frapaise, Christianne Bichouard, and Thierry Levayer (Etablissement Français du Sang, Montpellier, France). We thank Bernard Klonjkowski (Ecole Vétérinaire d’Alfort, Maissons-Alfort, France) for PAV-3 and BAV-3, Denis Ponard (Université Joseph Fourier, Grenoble, France) for advice in complement hemolytic testing, and Jean-Yves Cesbron (Université Joseph Fourier, Grenoble, France) for continuous encouragement. We thank Laure Pantel and Mireia Pellegrin (Institut de Génétigue Moleculaire de Montpellier, Montpellier, France) for help with the ELISA. We thank the members of our laboratories for constructive comments, suggestions, and help during the course of this study. The Association Française contre les Myopathies (AFM) and Vaincre les Maladies Lysosomales (VLM) supported this work, in part. E.J.K. is an Inserm Director of Research, and M.P. was an AFM/VML ­ fellow. The authors have no conflicting financial interests.

2006

Supplementary material Table S1. NAb, IgG, IgA and IgM titers. Table S2. NAb, class G, A, and M Ig titers versus TM proliferation. Table S3. NAb titers average and % responder for 50 donors. Table S4. CAV-2 capsid protein recognized and NAb titer. Table S5. NAb, class G, A, M immunoglubulin titers in plasma used for complement pathway activation. Figure S1. Principles of complement activation.

References

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