EP-4399515-B1 - METHOD FOR EVALUATING THE CAPACITY OF A SERUM TO NEUTRALISE A VIRUS

Inventors

• FRABONI, BEATRICE
• DECATALDO, Francesco
• TESSAROLO, Marta
• SCAGLIARINI, ALESSANDRA
• SAMBRI, Vittorio
• GIOVANNINI, CATIA

Dates

Publication Date

20260506

Application Date

20220908

Claims (12)

1. An in vitro method for evaluating the capacity of a serum to neutralise a virus, comprising the steps of: a) preparing a solution comprising at least one cell, at least one serum and at least one virus; b) placing the solution in contact with an organic electrochemical transistor comprising a source electrode, a drain electrode and a gate electrode, wherein the source and drain electrodes are electrically connected by means of a conductive channel comprising a conducting or semiconducting polymer, wherein the at least one cell is adhered to said channel and/or to the gate electrode or the at least one cell is adhered to a permeable porous support interposed between the conductive channel and the gate electrode; c) applying a potential difference between the drain electrode and the source electrode, applying a plurality of pulses of a potential difference between the gate electrode and the source electrode and measuring a respective plurality of values of pulses of current flowing through the channel; d) calculating a value of an estimation of a response time of the organic electrochemical transistor, as a function of the plurality of values of the measured current; e) comparing the value of the estimated response time with respect to a threshold value and detecting, as a function of said comparison, whether the serum has a neutralising capacity against the at least one virus.
2. The method according to claim 1, wherein the value of the estimated response time is calculated by interpolating the plurality of values of the measured current with a bi-exponential decay curve and measuring therefrom the value of a time response parameter of the bi-exponential curve.
3. The method according to claim 2, wherein the current I_d through the channel is calculated with the following formula: I _ d = a * exp t / T 1 + b * exp t / T 2 + e wherein: - t is the time; - exp is the exponential function; - a, b, e are configuration parameters; - T1 is a time parameter that represents the polymer charging time influenced by the ion blocking properties of the cell layer; - T2 is a time parameter that depends on the cell layer charging time; and wherein the current pulses are normalised between a value 1 which corresponds to the maximum channel current value under conditions of direct polarisation of the organic electrochemical transistor and a value 0 which corresponds to an average value of a defined number of points of the channel current when the latter has reached the stationary regime, and wherein the response time of the organic electrochemical transistor is equal to the value of the time parameter T1 over time normalised with respect to the value of T1 that it has prior to preparation of the solution.
4. The method according to any one of the preceding claims, wherein said plurality of pulses of the potential difference between the gate and source electrodes is a square wave having a defined difference between a high value and a low value, wherein the high value of the square wave is comprised between 0.1 Volts and 0.8 Volts and the low value of the square wave is comprised between -0.3 Volts and 0.5 Volts, in particular the potential difference between the high and low values of the square wave is equal to 0.3 Volts, wherein the duration of the high value of each pulse is comprised between 0.005 seconds and 10 seconds, in particular equal to 0.5 seconds, wherein the duration of the low value is comprised between 0.1 seconds and 30 seconds, in particular equal to 1.5 seconds, and wherein the value of the potential difference between the drain electrode and the source electrode is comprised between -0.4 Volts and 0.2 Volts, in particular equal to 0.1 Volts.
5. The method according to any one of the preceding claims, comprising, before step a), the steps of: - preparing a further solution comprising said at least one cell and said at least one serum; - placing the further solution in contact with the organic electrochemical transistor; - applying said potential difference between the drain electrode and the source electrode, applying said plurality of pulses of a potential difference between the gate electrode and the source electrode and measuring a respective further plurality of values of current flowing through the channel and through said at least one cell; - calculating said threshold value, as a function of the further plurality of values of the measured current.
6. The method according to any one of the preceding claims, wherein the conducting polymer is selected from: PEDOT:PSS, PEDOT-S, PEDOT:TOS, PEDOTOH:CIO4, PEDOT-co-PEDOTOH:CIO4, P3HT, PTHS, BBL, p(g2T- TT), PTHS-TMA<+>-co-P3HT, p(gNDI-g2T), p(g0T2-g6T2), Polyaniline, Polypyrrole and P-90.
7. The method according to any one of the preceding claims, wherein the at least one virus belongs to the Coronaviridae family.
8. The method according to claim 7, wherein the at least one virus is selected from SARS-CoV, MERS-CoV and SARS-CoV-2.
9. The method according to claim 7 or 8, wherein the at least one virus is SARS-Cov-2.
10. The method according to any one of the preceding claims, wherein the serum is obtained from an individual who has received a treatment against the at least one virus.
11. The method according to claim 10, wherein the at least one treatment is a vaccine against the at least one virus, preferably a vaccine against a Coronavirus, more preferably against SARS-CoV-2.
12. The method according to any one of claims 1-11, wherein the serum is obtained from an individual who has developed an infection caused by the at least one virus, preferably caused by a coronavirus, more preferably caused by SARS-CoV-2.

Description

TECHNICAL FIELD OF THE INVENTION The present invention generally relates to the field of in vitro analysis of cell cultures. More in particular, the present invention concerns a method for evaluating the capacity of a serum to neutralise a virus. PRIOR ART The growing spread of infectious diseases, in particular, of infectious diseases of the respiratory tract, is driving scientific research towards a search for new diagnostic and therapeutic strategies. The COVID-19 pandemic caused by the coronavirus (CoV) SARS-CoV-2 has led to an unprecedented demand for diagnostic tests and therapies against the virus, aggravating an epidemiological situation already abounding in new potential pathogenic microorganisms. Various studies conducted on severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) have demonstrated the presence of specific neutralising antibodies against these viruses in 80-100% of patients two weeks after the appearance of symptoms. The antibodies can suppress the replication of SARS-CoV-2 through viral neutralisation, but they can also participate in the pathogenesis and progression of COVID-19 through a process defined as antibody-dependent enhancement. For this reason, it is very important to evaluate the presence of neutralising antibodies, produced during SARS-CoV-2 infection, for prognostic purposes in clinical practice. Furthermore, the immunological tests available for quantifying the neutralising antibodies in patients are fundamental also in order to provide information and data on the effectiveness of a vaccine, with the aim of giving priority to different vaccine candidates and supporting the requests for authorisation of the vaccine. Harmonisation of the results of tests on neutralising antibodies (both against SARS-CoV-2 and against pseudoviruses) is particularly important also in preclinical studies on vaccines, which involve non-human primates, to enable accurate comparisons with those obtained in clinical studies. There exist various methods for determining the antibody concentration, but the PRNT (plaque reduction neutralisation test) is considered the gold standard for measuring the levels of neutralising antibodies. The PRNT was developed by Dulbecco for animal virus inactivation (or neutralisation) studies and modified to measure the antibody neutralisation titre in serum. The PRNT has already been used to test the level of protection of the population in countries affected by other zoonotic coronaviruses, such as MERS-CoV. The PRNT requires the formation of a virus-antibody complex in vitro, with an incubation of 30 minutes at 37°C and subsequent seeding on cells susceptible to viral infection. After the incubation (typically 72 hours), the cytopathic effect (CPE) is evaluated by fixing and staining the cells with formaldehyde and crystal violet and, subsequently, reading the absorbance with spectrophotometer at 560 nm. The highest serum dilution that neutralises 90% of viral replication and, therefore, its cytopathic effect, is reported as the neutralising titre. This technique entails many economic costs, from the materials used to the highly specialised personnel necessary to the disposal of toxic waste. Furthermore, it requires a long time to obtain the results (72 hours after infection), thus precluding rapid reporting times. Furthermore, formaldehyde and crystal violet are known to have carcinogenic effects and must be disposed of according to very strict regulations. Because of the high infectivity and pathogenicity of SARS-CoV-2, the virus must be handled in specific biosafety level 3 (BSL-3) facilities. The PRNT is thus technically challenging and difficult to automate, making it unsuitable for large-scale studies on serum samples, such as phase III clinical studies on a human vaccine. In order to avoid having to do with infectious CoV-2, various safe systems have been developed, which are based on pseudoviruses at a biosafety level of 2 (BSL2). Even though they have demonstrated to be sensitive and reliable, said tests are often cumbersome, long and costly in procedural terms. Thus, there is a strongly felt need to have a test capable of evaluating the presence of antibodies in a serum, i.e. the neutralising capacity of a serum. In particular, there is a great demand for a test or a method that enables the presence of antibodies in a serum and the capacity of the latter to neutralise the virus to be evaluated rapidly and with limited costs. Di Tian et al., "Novel, Real-Time Cell Analysis for Measuring Viral Cytopathogenesis and the Efficacy of Neutralizing Antibodies to the 2009 Influenza A (H1N1) Virus", PloS ONE, vol.7, issue 2, page e31965, discloses a real-time cell analysis system to measure the cytopathic effect of the 2009 influenza A (H1N1) virus and the efficacy of neutralizing antibodies in human sera to this virus. Francesco Decataldo et al., "Organic Electrochemical Transistors: Smart Devices for Real-Time Monitoring of Cellu