WO2004102215A1

WO2004102215A1 - Assay for testing neurotoxin vaccine efficacy

Info

Publication number: WO2004102215A1
Application number: PCT/US2004/010392
Authority: WO
Inventors: Joseph J. Pancrazio; Guenter W. Gross; David E. Steele
Original assignee: The United States Of America, As Represented By The Secretary Of The Navy Naval Research Laboratory
Priority date: 2003-05-07
Filing date: 2004-03-29
Publication date: 2004-11-25

Abstract

An assay to test the efficacy of neurotoxin vaccines comprising a neuronal network having a baseline spike or burst rate of activity, a neurotoxin that inhibits the spike or burst rate when added to the neuronal network, and an antiserum that neutralizes the neurotoxin. The efficacy of the vaccine is determined by comparing the baseline spike or burst rate and the spike or burst rate inhibition after the antiserum and neurotoxin are added to the neuronal network. A low spike or burst rate inhibition indicates an effective antiserum. Also disclosed is the related method of testing the efficacy of a neurotoxin vaccine.

Description

ASSAY FOR TESTING NEUROTOXIN VACCINE EFFICACY

Technical Field

The present invention relates to assays and, more specifically, to assays that test the efficacy of vaccines.

Background Art

The cause of paralysis in the food poisoning disease known as botulism is a neurotoxin produced as distinct serotypes (types A, B, C, D, E, F and G) by particular strains of Clostridium botulinum. To induce paralysis, the toxin binds rapidly and strongly to presynaptic cholinergic nerve terminals and inhibits the exocytosis of acetylcholine by decreasing the frequency of acetylcholine .release. While the botulinum toxins act on the peripheral nervous system to produce paralysis and lethality, these neurotoxins also can inhibit synaptic transmission in a variety of neuronal -cell types under culture conditions. Since botulinum toxins are considered threats as biological weapons to military personnel and civilian populations, there has been a substantial effort to develop vaccines for the various botulinum toxin serotypes, in particular botulinum toxins A and B. Testing the efficacy of botulinum toxin vaccines requires the assessment of the antiserum, serum that contains antibodies against the neurotoxin antigen generated via the vaccination process. The present method for testing antisera, and thus vaccine efficacy, is to combine live botulinum toxin with the antiserum and inject the combination into mice. Varying the concentration ratio of antiserum and botulinum toxin and quantitating mice lethality developed after 2-3 days of injection permits a comparison of vaccine protocol efficacy. Clearly, the more effective vaccine protocols generate more potent antisera capable of neutralizing the botulinum toxin as assessed by mice lethality.

Extracellular potentials from excitable cells result from the generation and propagation of all-or-nothing action potentials or spikes. A collection of spikes is a burst. In the nervous system, these spikes play a critical role in triggering neurotransmission, where the rate of spike firing or occurrence is a fundamental measure of neuronal function. While extracellular spike amplitudes can be two to three orders of magnitude smaller than those assessed directly (intracellular or whole cell patch clamp measurements), extracellular recordings have the advantageous characteristics of being non-invasive or non-damaging to the cells and, therefore, amenable to long-term assays (hours and days) compared to intracellular recording (minutes). In addition, extracellular recording is conducive for multielectrode analysis where excitable cells are cultured on or near microelectrode contacts, such that simultaneous measurements from a network of cells can be readily accomplished. For these reasons, cultured excitable cells have been proposed as a basis for an assay or biosensor operation, whereby changes in spontaneous or evoked extracellular potentials reflect physiologic changes associated with toxicant or drug exposure. See G. W. Gross, A. Harsch, B. K. Rhoades & W. Gopel, "Odor, drug and toxin analysis with neuronal networks in vitro: extracellular array recording of network responses," BIOSENS BIOELECTRON 1997 A, 12:373-393; G. W. Gross, S. Norton, K. Gopal, D. Schiffrnann & A. Gramowski, "Nerve cell network in vitro: Applications to neurotoxicology, drug development, and biosensors," CELLULAR ENGINEERING 1997B, 2:138-147; and J. J. Pancrazio, J. P. Whelan, D. A. Borkholder, W. Ma & D. A. Stenger, "Development and application of cell- based biosensors," ANN. BIOMED. ENGIN. 1999, 27:1-15. Great progress has been made in growing nerve cell networks over substrate-integrated thin film multielectrode arrays derived from primary, embryonic cell isolations from a variety of nervous system tissue types including spinal cord and frontal cortex. The resulting cultures have distinct spike or burst rate characteristics, show great longevity and stability reaching 6 months, and have pharmacological profiles that are consistent with-the tissue from which they are derived. See E.'W. Keefer, A. Gramowski, D. A. Stenger, J. J. Pancrazio & G. W- Gross, "Characterization of acute neurotoxic effects of trimethylolpropane phosphate via neuronal network biosensors," BIOSENSORS & BIOELECTRONICS, 2001, 16, 513-525; E. W. Keefer, A. Gramowski & G. W. Gross, "NMDA receptor-dependent periodic oscillations in cultured spinal cord networks," J. NEUROPHYSIOL, 2001, 86, 3030-3042; G. W. Gross, B. K. .Rhoades & R. S. Jordan, "Neuronal networks for biochemical sensing," SENSORS ACTUAT B 1992, 6:1-8; G. 'W. Gross, "Internal dynamics of randomized mammalian neuronal networks in culture," ENABLING TECHNOLOGIES FOR CULTURED NEURAL NETWORKS, D. A. Stenger. & T. M. McKenna, eds., New York, Academic Press, 1994, pp. 277-317; G. W. Gross, A. Harsch, B. K. Rhoades & W. Gopel, "Odor, drug and toxin analysis with neuronal networks -in vitro: extracellular array recording of network responses," BIOSENS BIOELECTRON 1997A, 12:373-393; and G. W. Gross, S. Norton, K. Gopal, D. Schiffrnann & A. Gramowski, "Nerve cell network in vitro: Applications to neurotoxicology, drug development, and biosensors," CELLULAR ENGINEERING 1997B, 2:138-147.

Disclosure of Invention

An assay to test the efficacy of neurotoxin vaccines is provided which includes a neuronal network having a baseline spike or burst rate of spontaneous neuronal activity, a neurotoxin that inhibits the spike or burst rate when added to the neuronal network, and an antiserum that neutralizes the neurotoxin. The efficacy of the vaccine is determined by comparing the baseline spike or burst rate and the spike or burst rate inhibition after the antiserum and neurotoxin are added to the neuronal network. A low spike or burst rate inhibition indicates an effective antiserum. The present invention results in at least two major advantages for assessing botulinum toxin vaccine efficacy. First, the present gold-standard technique involves the use of living mice, whereby quantitation is based on population responses to generate relative lethality curves. Since animal experiments require veterinary staff and rely on binary metrics (live/dead), such approaches are expensive and exhibit great variability, and thus require an extremely large number of replicates. Instead, the use of cultured neuronal networks can generate a dose- response curve, thus substantially reducing the number of experiments that need to be performed to evaluate relative vaccine efficacy. Tissue from a single culture of embryonic tissue (1 pregnant mouse dam) results in 12 embryos, thus seeding 100 cultured neuronal networks suitable for pharmacological screening. This result translates into a major reduction in the cost associated with animal experiments. Second, mice lethality assays require monitoring over the course of 2-3 days, consistent with the .pharmacokinetics of the neurotoxin in organisms. Like many in vitro assays, the .cultured neuronal networks respond much <more quickly to the pharmacological agents such that the -time required for vaccine evaluation can be reduced to 3-6 hours.

Brief Description of Drawings

These and other objects, features and advantages of the invention, as well as the invention itself, will become better understood by reference to the following detailed description, appended claims, and accompanying drawings where: Fig. 1 shows the inhibition in the spike and burst rates of spontaneous neuronal activity after botulinum toxin is added to a neuronal network; and

Fig. 2 shows the inhibition in the spike rate of spontaneous neuronal activity after antiserum and botulinum toxin are added to a neuronal network. Best Mode for Carrying Out the Invention An assay to test the efficacy of neurotoxin vaccines according to a preferred embodiment of the present invention uses a neuronal network to monitor the change in activity after a neurotoxin and an antiserum are added to the network. In general, neuropharmacological analysis can be accomplished using cultured neuronal networks grown over microelectrode arrays by monitoring spike or burst activity at multiple microelectrode contacts. Quantification of network activity can be performed by considering microelectrode contacts or after applying pattern recognition to separate waveforms from the same microelectrode contact that represent the activity of multiple, distinct neurons or units. The spike or burst rate is quantified by detecting the spikes or bursts at each microelectrode contact or unit in time bins that are typically 1 minute in duration. The mean spike or burst rate for the network is the ensemble average of the spike or burst rates in the same time bin across all microelectrode contacts or units. The neuronal network has a baseline spike or burst rate of activity. Adding a neurotoxin such as botulinum toxin or tetrodotoxin to the network will inhibit the mean spike or burst rate. If both an antiserum and a neurotoxin are added to the network (either sequentially or following titration of antiserum to toxin prior to addition to the network), some of the toxin will bind to the antiserum and some of the toxin will remain free. The efficacy of the antiserum is determined by measuring how much free toxin is present. This can be done by comparing the baseline spike or burst rate to the spike or burst rate after the antiserum and the neurotoxin are added. A -low decrease in the spike or burst rate indicates an effective antiserum. A preferred embodiment of the present invention utilizes the activity derived from neuronal networks cultured over multi-microelectrode arrays as a basis for neurotoxin vaccine testing. Spontaneous neuronal activity is measured -under control conditions to establish a baseline spike or burst rate over all active channels. In the absence of neurotoxin or pharmacological treatment, the derived spike or burst and burst rates are stable and consistent for periods reaching 2 months.

The techniques used to fabricate and prepare microelectrode arrays (MEAs) have been described in detail in G. W. Gross, "Simultaneous single unit recording in vitro with a photoetched laser deinsulated gold multi-microelectrode surface," IEEE TRANS BIOMED ENGIN 1979, 26:273-279 and G. W. Gross, W. Wen, & J. Lin, "Transparent indium-tin oxide patterns for extracellular, multisite recording in neuronal cultures," J. NEUROSCI METH 1985; 15:243-252. Although various electrode conductor patterns ;have been described, most data have been derived from recording matrices consisting of 32-64 microelectrodes. See G. W. Gross, "Internal dynamics of randomized mammalian neuronal networks in culture," ENABLING TECHNOLOGIES FOR CULTURED NEURAL NETWORKS, D. A. Stenger & T. M. McKenna, eds., New York, Academic Press, 1994, pp. 277-317. MEA surfaces are activated by flaming and coated with poly-D-lysine and laminin. See J. H. Lucas, L. E. Czisny & G. W. Gross, "Adhesion of cultured mammalian CNS neurons to flame-modified hydrophobic surfaces," IN VITRO 1986; 22:37-43. Dissociated tissue cultures are prepared according to the basic method established by B. R. Ranson, E. Neale, M. Henkart, P. N. Bullock & P. G. Nelson, "Mouse spinal-cord in cell-culture 1. Morphology and intrinsic neuronal electrophysiologic properties," J. NEUROPHYSIOL 1977; 40:1132-1150. Embryonic nervous system tissue is dissociated enzymatically and mechanically seeded on the in cell media with 5% horse serum onto MEA surfaces. See G. W. Gross, "Internal dynamics of randomized mammalian neuronal networks in culture," ENABLING TECHNOLOGIES FOR CULTURED NEURAL NETWORKS, D. A. Stenger & T. M. McKenna, eds., New York, Academic Press, 1994, pp. 277-317. Spontaneous electrical activity from microelectrode sites is apparent after approximately 7 days in the form of random spiking and stabilized in terms of regular patterns by 15 days in vitro.

In a preferred embodiment, MEAs are placed into constant-bath recording chambers and maintained at 37°C while the pH is held at 7.4 with a continuous stream of filtered, humidified, 10% CO2 in air. Activity is recorded by a multi-channel amplifier system consisting of 64 two- stage amplifiers with a total system gain of approximately 8,000-10,000, although reliable neuropharmacological assessments of toxin activity can be achieved with as few as 10 active microelectrode contacts or units per network. Spike or burst rates can ^"be quantified on-line as a measure of neuronal network activity. Mean spike or burst rates vary from culture-to-culture where neuropharmacological analysis can be readily performed for units or microelectrode contacts showing rates exceeding 1 -Hz. Addition of bicuculline or other GABA_A receptor antagonists to -the neuronal network to excite and/or regularize spike or burst activity diminishes neither the network sensitivity to botulinum toxin nor the inhibitory effects of antisera on botulinum toxin. The present invention can be used to evaluate vaccine efficacy for a variety of neurotoxins, such as botulinum toxins, tetrodotoxin and tetanus toxin, where vaccines have been contemplated or sought. In a preferred embodiment, the neurotoxin is botulinum toxin. When botulinum toxin is added to a neuronal network, the spike or burst rate decreases until there are virtually no action potentials detectable (however, some cells spontaneously fire in the absence of synaptic input). The rate of spike or burst frequency inhibition is concentration-dependent, such -that higher concentrations reach complete neuronal network inhibition faster than lower concentrations. As shown in Fig. ,1 , the addition of botulinum toxin to a neuronal network induces a marked inhibition in the measured spike and burst rates after a latency period of approximately 2 hours. The toxins can be added as a lOOx or greater concentrated bolus in H O at a volume not exceeding l/100^th of the media in the recording chamber. Alternatively, the appropriate concentration of the toxin can be suspended in bathing media at its final concentration and a complete media exchange can -be performed by passing the volume equivalent to 4x the media in the recording chamber, typically accomplished via a peristaltic pump at a rate of not more than 1 ml/min. Toxins are applied only after a steady baseline of mean spike or burst rate for at least 45 minutes has been demonstrated. Spike or burst rate inhibition is readily detected by a decrease in the number of spikes or bursts per unit time, and is quantitatively displayed by monitoring mean spike or burst rate over the 3-6 hour duration of the assay. The typical baseline coefficients of variation of the mean spike or burst rate is less than 10% such that changes of 20% are readily apparent and significant. Since the neurons respond only to free, functionally active levels of the neurotoxin, the methodology for vaccine efficacy involves co-application of antiserum and botulinum toxin to the cultured networks. The antiserum can be added before the toxin or simultaneously with the toxin. Experiments have shown that application of control human serum after dialysis to 5 eliminate physiologic neuroactive constituents such as glutamate and glycine does not alter cultured neuronal network spike or burst rates. After only 3-6 hours, the degree of spike or burst rate inhibition corresponds to the free, functional concentration of botulinum toxin remaining after neutralization. Those antiserum that exhibit the highest potency for botulinum toxin neutralization will induce the least inhibition of spike or burst frequency or yield the slowest

1.0 decay in spike or burst frequency. Fig. 2 shows the inhibition in the spike rate of spontaneous neuronal activity after antiserum and botulinum toxin are added to a neuronal network. A volume of antiserum at a volume such that the final concentration -does not exceed 5% of the media recording volume is suspended in recording media and applied to the neuronal network 30 minutes prior to challenge with a fixed concentration of neurotoxin (or antiserum toxin is titrated

15 against a fixed quantity of toxin prior to addition to the neuronal -network). In practice, antisera efficacy is envisioned to be accomplished by processing at least n number of networks in parallel, perhaps occupying the same microelectrode array but separated into distinct wells. Networks 1 through n-1 would be treated with low, medium, and high antisera concentrations respectively, whereas the .nth network would receive no- antisera protection to ensure that the

20 toxin is functional. -Each of the wells would then be challenged with the same concentration of neμrotoxin and efficacy based on the lowest antisera concentration required to provide protection in terms of -little or no change in mean spike or burst rate of the network.

The approach described herein can also be used to identify strains of botulinum toxin from serum samples of potentially poisoned individuals. With this application, a 1 ml volume of

25 potentially contaminated serum is combined with 0.25 ml of a known antitoxin distributed by the Centers for Disease control and Prevention that is specific for one of the botulinum toxin serotypes and has a well-defined level of efficacy. See "Botulism in the United States, 1899- 1996" U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. The combined antitoxin and botulinum toxin exposed serum sample would then be

30 applied to cultured neuronal networks not exceeding a final concentration of 5% of the recording chamber media. In practice, each of the antitoxin possibilities would be surveyed simultaneously to identify the strain of botulinum toxin. The network that fails to show a reduction in mean spike or burst rate would correspond to a successful protection by a specific antitoxin and thus identify the strain of the botulinum toxin. The approach described herein is amenable to massively parallel analysis, e.g. 96- or 384- well format assays, where many neurotoxin-antiserum combinations could be assayed simultaneously. Further, this approach for neurotoxin testing need not depend exclusively on primary neurons derived from mice; this approach depends only of the ability of the cultured cells to generate synaptic contacts. Progress in neural stem cell technology suggests that this approach for vaccine testing could be performed with neuronal networks derived from stem cells of a variety of species including humans, thus further reducing the requirements for animals.

The above description is that of a preferred embodiment of the invention. Various modifications and variations are possible in light of the above teachings. It is therefore to be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described. Any reference to claim elements in the singular, e.g. using the articles "a," "an," "the," or "said" is not construed as limiting the element to the singular.

Claims

1. An assay to test the efficacy of a neurotoxin vaccine, comprising: a neuronal network having a baseline spike or burst rate of spontaneous neuronal activity; a neurotoxin that inhibits the spike or burst rate when added to the neuronal network; and an antiserum that neutralizes the neurotoxin; wherein the efficacy of the vaccine is determined by comparing the baseline spike or burst rate to the spike or burst rate after the antiserum and the neurotoxin are added to the neuronal network, wherein a low spike or burst rate inhibition indicates an effective antiserum.

2. The assay of claim 1 wherein said neurotoxin is botulinum toxin.

3. The assay of claim 1 wherein said neurotoxin is botulinum toxin serotype A.

4. The assay of claim 1 wherein said neuronal network is cultured over a multi- microelectrode array.

5. The assay of claim 1 wherein said antiserum can be added to said neuronal network before said neurotoxin or simultaneously with said neurotoxin.

6. A method to test the efficacy of a neurotoxin vaccine, comprising the steps of:

(a) establishing a baseline spike or burst rate of spontaneous neuronal activity of a neuronal network; (b) adding an antiserum and a neurotoxin to the neuronal network;

(c) comparing the baseline spike or burst rate to the spike or burst rate after the antiserum and the neurotoxin are added wherein a low spike or burst rate inhibition indicates an effective antiserum.

7. The method of claim 6 wherein said neurotoxin is botulinum toxin.

8. The method of claim 6 wherein said neurotoxin is botulinum toxin serotype A.

9. The method of claim 6 wherein said neuronal network is cultured over a multi- microelectrode array.

10. The method of claim 6 wherein said antiserum can be added to said neuronal network before said neurotoxin or simultaneously with said neurotoxin.