WO2022053944A1 - Immunoassay for the detection of antibodies against merkel cell polyomavirus (mcpyv) using synthetic peptides - Google Patents

Abstract

An in vitro assay method for the detection of antibodies directed against Merkel cell polyomavirus (MCPyV) in a sample of a subject's biological fluid and the related kit are described. The method and kit of the invention are based on the use as a capture agent of one or more isolated peptides characteristic of Merkel cell polyomavirus (MCPyV), which do not cross-react with other polyomaviruses.

Description

Immunoassay for the detection of antibodies against Merkel cell polyomavirus (MCPyV) using synthetic peptides

This invention is within the field of immunodiagnostics.

More particularly, this invention relates to an in vitro immunoassay, preferably an indirect ELISA, wherein synthetic peptides are used as viral antigens for the identification of antibodies directed against Merkel cell polyomavirus (MCPyV).

Merkel cell polyomavirus is one of 14 human polyomaviruses known to date (1, 2). In 2008, its genome was identified, integrated into the cellular DNA of a Merkel cell carcinoma (MCC), a rare human skin cancer (3). MCPyV, like other human polyomaviruses, e.g., BKPyV and JCPyV, is ubiquitous, i.e., it is widely distributed in the human population (1, 2). Polyomaviruses generally do not cause disease in healthy subjects, and although various viruses are associated with disease in immunocompromised hosts, MCPyV is the only one directly associated with cancer (3). In fact, MCPyV has been classified by WHO/IARC as probably carcinogenic to humans (group 2A) (1, 2)

Merkel cell carcinoma (MCC), although a rare skin cancer, is very aggressive. MCC has a high mortality rate, approximately 46% over 5 years (3). It has an incidence of l/~3 million cases per year in Europe. This cancer is common among white and elderly individuals (>60 years of age) with excessive UV exposure. The oncogenic DNA virus, MCPyV, is its main etiologic agent. In fact, MCPyV DNA is present in approximately 80% of all MCCs (3). Anti-MCPyV antibodies are present in nearly 100% of patients with MCC, in 60-80% of healthy adults, and in 35% of children 1-5 years of age (4). These data indicate that MCPyV is ubiquitous. MCPyV DNA has been detected in the leukocyte-platelet layer and in sera from healthy adults with a prevalence of 22% and 2.6%, indicating that MCPyV may replicate in healthy subjects, reactivating from its latent phase (5, 6). In fact, after the primary infection that occurs in pediatric age, MCPyV remains latent in immunocompetent hosts, until a weakening of the immune system leads to viral reactivation and integration of viral DNA into the human genome, resulting in permanent expression of the two viral oncoproteins, Large T (LT) and Small T (ST) antigens. Similarly, it is known that reactivation and consequent infection of human polyomaviruses (3), including JCPyV and BKPyV, may be induced by immune deficiency.

In immunocompromised hosts, the risk of occurrence of MCC increases (7); in fact, approximately 10% of patients affected by MCC show immune deficits. Immunosuppressed patients are also more prone to develop virus-associated cancers, including Kaposi’s sarcoma and Burkitt’s lymphoma, which are associated with HHV8/ KSHV and EBV herpesviruses, respectively.

For scientific research purposes, some groups, including that of the inventors, have employed techniques of molecular biology, such as both qualitative and quantitative polymerase chain reaction (PCR) methods, to detect the presence of MCPyV DNA sequences in human samples (7, 8). However, there are currently no specific, rapid, and inexpensive methods commercially available using immunologic techniques to test for the presence of antibodies to MCPyV in humans. In fact, current immunological techniques reported in the scientific literature are based on the use of “virus-like particles” (VLPs), aggregates of recombinant VP1 of MCPyV that self-assemble in vitro (9). VLPs as viral antigens are used for anti-MCPyV antibody detection. The production of VLPs involves several laborious steps, including in vitro synthesis from Spodoptera frugiperda cells, or other recombinant bacterium, isolation, quantification, and finally the use of ELISA assays to verify the presence of antibodies against MCPyV (9, 10). Alternatively, more recent studies report the development of ELISA methods based on multiple antibody detection called “Multiplex Serology” based on the use of fluorescent microspheres in combination with glutathione S-transferase (GST). These systems, although innovative since they allow the simultaneous detection of different viruses, have various methodological limitations as they are still based on the use of VLPs or soluble recombinant proteins (10, 11). In fact, it is important to point out that the use of recombinant VLPs could increase the probability of cross -reaction between different viruses that, although different, present a certain degree of homology, and therefore invalidate the result obtained (9, 10, 11).

Given the current state of knowledge, the importance of having standardized, specific, sensitive, rapid, and inexpensive analysis methods that allow to unequivocally identify the presence of antibodies against MCPyV, both in the sera of patients affected by different diseases, including neoplasms such as MCC, and in healthy individuals, for example blood, stem cell, and organ donors, is evident.

Immunologic surveillance for MCPyV in patients treated with monoclonal antibodies, such as patients with multiple sclerosis and autoimmune diseases, such as rheumatoid arthritis and ankylosing spondylitis (7), is also of primary interest. In fact, prior data from the inventors indicate an approximately 1,000-fold increase in the incidence of MCC in patients with autoimmune diseases undergoing therapy with biologic drugs and monoclonal antibodies. For this purpose, monitoring the antibody levels against MCPyV in the sera of such patients over time, with a standardized, sensitive, rapid, and inexpensive method, is of primary importance to ensure a favorable outcome (7).

These and other needs are satisfied by the present invention, which provides the immunological method and related kit defined in the appended independent claims, suitable for the detection of anti-MCPyV antibodies in a sample of a biological fluid from a subject or patient to be analyzed.

Further features of the invention are identified in the dependent claims, which form an integral part of the description.

The present invention makes it possible to overcome the lack of specific immunological analytical methods through the use of a series of new synthetic peptides, used as viral antigens in an immunoassay, which allow the detection of antibodies against MCPyV. In particular, the inventors have identified unique short peptide sequences in MCPyV that represent specific epitopes and selective immunological targets for antibodies present in the sera and other fluids from MCPyV -infected subjects. Thus, a first aspect of the present invention is an isolated peptide, corresponding to a specific epitope or antigen of a viral capsid protein 1, 2, or 3 (VP1, VP2, and VP3) of Merkel cell polyomavirus (MCPyV), consisting of the amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2. Such an isolated peptide is characteristic of MCPyV, in the sense that it does not cross-react with other polyomaviruses.

A second aspect of the present invention is an in vitro method for detecting anti-MCPyV antibodies in a sample of a biological fluid from a subject, comprising the steps of: a) contacting said sample with a capture agent comprising one or more isolated MCPyV peptides as defined above (i.e., SEQ ID NO:1 and/or SEQ ID NO:2); and b) detecting the formation of an immunocomplex between anti-MCPyV antibody/antibodies and said capture agent, wherein the formation of the immunocomplex is indicative of the presence of anti-MCPyV antibodies in the biological fluid sample.

In a preferred embodiment, the anti-MCPyV antibodies possibly present in the biological fluid sample are human IgG and/or IgM antibodies. In this case, the formation of the immunocomplex is revealed by using an anti-human secondary antibody.

In another preferred embodiment, the biological fluid to be analyzed is selected from the group consisting of serum, plasma, blood, saliva, urine, or cerebrospinal fluid. Among these, serum is most preferred.

In yet another preferred embodiment, the immunocomplex is detected by immunoprecipitation, immunoassay (preferably ELISA or RIA or fluorescence immunoassay), western blot, FACS analysis or an immunocytochemical/immunohistochemical technique. Among these, an indirect ELISA assay is most preferred.

A third aspect of the present invention is a diagnostic kit for determining the presence of anti-MCPyV antibodies in a sample of a biological fluid from a subject, the kit comprising (i) a capture agent comprising one or more isolated peptides as defined above, and (ii) a reagent for detecting the immunocomplex possibly formed between the anti-MCPyV antibodies and the capture agent. The use of an anti-human secondary antibody is preferred as the reagent for detecting the presence of the immunocomplex.

The diagnostic kit of the invention may further comprise a solid assay support, such as an ELISA plate.

The following experimental section is provided by way of illustration only and is not intended to limit the scope of the invention as defined by the appended claims.

section

Materials and methods

Biological samples

Sera from normal individuals, males and females of different ages, were provided by several university clinics, hospitals, and research centers, after signing informed consent in accordance with the Helsinki Declaration. The Ferrara Ethics Committee approved the study, ID: 151078 (7).

Peptides

The structural or capsid proteins of polyomaviruses, toward which antibodies are produced, are encoded by the tardive region of the viral genome. The peptides of the invention were synthesized using standard techniques and equipment (12). The two synthetic peptides of the invention, used as MCPyV- specific antigens in an immunoassay for detecting antibodies against MCPyV in sera (preferably an indirect ELISA assay), have the following amino acid sequences:

Peptide MCPyV- VP1 S (PEP-S) (24 amino acids):

NH2- NSPDLPTTSNWYTYDLQPKGSS -COOH (SEQ ID NO:1) and

Peptide MCPyV- VP2/VP3 F (PEP-F) (25 amino acids): NH2- SLSPTSRLQIQSNLVNLILNSRWVF -COOH (SEQ ID NO:2)

The amino acid sequences of the PEP-S and PEP-F peptides are located within the coding sequences for the tardive MCPyV genes: the VP 1, VP 2, and VP 3 proteins (Fig. 1). Although the gene regions of MCPyV VP1 and VP2/VP3 exhibit some degree of homology with other polyomaviruses, including JCPyV, BKPyV, and SV40, bioinformatic analyses carried out by the inventors revealed that the genomic regions corresponding to PEP-S and PEP-F advantageously exhibit extremely low homology when compared to other polyomaviruses.

Identification of antibodies against MCPyV in human sera by indirect ELISA immunoassay with synthetic peptides used as viral antigens

The assay method comprises several steps, as previously reported (13-15).

1. Coating. Lyophilized peptides were dissolved in Coating Buffer (Candor Bioscience GmbH - CABRU s.a.s. Milan) lx until reaching a concentration of 100 pg/ml, aliquoted and stored at -20°C until the moment of use. Plates with 96 wells were used wherein the synthetic peptide was adhered in a step termed “coating.” The peptides were diluted 1:2 in IX coating buffer, resulting in a final concentration of 50 pg/ml. In each well, 100 pl of the peptide thus prepared was added using a multichannel pipette, the plate was allowed to incubate in the dark at 4°C for 16-18 hours in order for the peptide to adhere properly to the bottom of the well of the plate. After the incubation period at 4°C, three washes were performed with 250 pl per well of Washing Buffer (Candor Bioscience GmbH - CABRU s.a.s. Milan) diluted 0.5x in double-distilled water, starting with a lOx stock concentration. A washer (Thermo Elctron Corporation, Wellwash 4 MK 2. Pobbiano, Italy) was used to perform the washes, aimed at removing excess peptide.

2. Blocking. This step involves the addition of the Blocking Solution (Candor Bioscience GmbH - CABRU s.a.s. Milan), which is able to complete the saturation of each well, in case there are areas not covered by the peptide. This solution prevents interaction between the primary antibody that may be present in the serum samples and the surface of the plate. 200 pl of blocking solution was added to each well with a multichannel pipette, and the plate was incubated for 90 minutes at 37°C in the dark. Subsequently, three washes were performed with 250 pl of Washing Buffer for each well, again using the washer.

3. Primary antibodies. Once the washes were performed, the serum samples to be assayed were placed in the corresponding wells. In this way, it is possible to check for the presence of any antibodies against MCPyV in the serum. The sera were diluted 1:20 in Low Cross Buffer (Candor Bioscience GmbH - CABRU s.a.s. Milan, Italy); 100 pl of serum thus diluted was aliquoted into each well. Each sample was analyzed in duplicate. The plate was then incubated at 37°C for 90 minutes in the dark. In each experiment there is also a positive control represented by serum from: (i) patients with Merkel cell carcinoma (MCC) who previously tested positive for IgG antibodies to MCPyV (7); (ii) sera from healthy individuals who previously tested positive for IgG antibodies to MCPyV (9). In addition, three controls found to be negative in previous experiments were included, which are used to identify the threshold value (cut-off).

4. Secondary antibody. Three washes with 250 pl of Washing Buffer were performed for each well to remove residual serum. A peroxidase-conjugated goat anti-human secondary antibody (CalbioChem, Milan), diluted 1:10,000 in Low Cross Buffer, was used. 100 pl of secondary antibody was then added to each well, using a multichannel pipette. It was allowed to incubate for 90 minutes at room temperature in the dark.

5. Spectrophotometer detection and reading. Three washes with 250 pl of Washing Buffer were performed for each well to remove the residual secondary antibody. To perform a spectrophotometer reading which is able to quantify the level of antibodies that may be present in the serum, 100 pl of 2,2’-Azino-bis (3 -ethylbenzothiazoline-6- sulfonic acid, ABTS; Sigma, Milan) was added to each well using a multichannel pipette. The plate was incubated at room temperature for 45 minutes in the dark. The reading was taken using a spectrophotometer (Thermo Elctron Corporation, Wellwash 4 MK 2. Pobbiano, Italy) at a wavelength of 405 nm. The results obtained were analyzed to evaluate the range of optical density (OD) obtained, checking that the values of the duplicates in the same plate had values which are comparable to each other. 6. Cut-off determination. The cut-off value was determined as described previously (13-15). Specifically, the cut-off was determined for each experiment using the ODs of the negative controls inserted in the plate. The threshold value was calculated by the average of three negative controls to which the triple standard deviation (+3D) was added. This method is consistent with previous experiments and the international literature (13-15). The cut-off value is calculated in each experiment and varies depending on the peptide used.

At the end of the analysis, a sample is considered positive for MCPyV if it is positive for both the MCPyV-VPl S peptide and the MCPyV-VP2/VP3 F peptide, thus having OD values higher than the threshold values of the individual experiments.

Results

Specificity assay of two synthetic peptides, PEP-S and PEP-F, used as viral antigens to reveal the presence of anti-MCPyV antibodies in human serum

In an indirect ELISA assay, a total of n=1080 serum samples were tested for the presence of antibodies against MCPyV. Specifically, samples were represented by sera from healthy individuals (n=l,080), stratified by age: <1 to 4 years (n=128), 5 to 10 years (n=54), 11 to 17 years (n=124), 18 to 30 years (n=130), 31 to 40 years (n=120), 41 to 50 years (n=141), 51 to 65 years (n=157), 66 to 70 years (n=40), 71 to 75 years (n=46), 76 to 80 years (n=57), 81 to 85 years (n=47), >86 years (n=36).

Prevalence of anti-MCPyV antibodies determined by indirect ELISA assay

With the indirect ELISA assay using PEP-S and PEP-F peptides, only sera that were simultaneously positive for both MCPyV-VPl S and MCPyV- VP2/VP3 F peptides used in the assay were considered MCPyV-positive. The prevalence of anti-MCPyV antibodies was 55.3% (597/1080) overall. Prevalence data of anti-MCPyV antibodies in subgroups of healthy individuals stratified by age are shown in Table 1. Specifically, the lowest prevalence of seropositivity, 11.7% (15/128), was found in the group of individuals <1-4 years. In fact, the difference in prevalence of anti-MCPyV antibodies in the group of individuals <1-4 years was statistically lower than in the groups of 5-10 years (48.1%, 26/54), 11-17 years (55.6%, 69/124), 18-30 years (63.1%, 82/130), 31-40 years (56.7%, 68/120), 41-50 years (64.5%, 91/141), 51-65 years (66.2%, 104/157), 66-70 years (62.5%, 25/40), 71-75 years (71.7%, 33/46), 76-80 years (64.9%, 37/57), 81-85 years (63.8%, 30/47),

>86 years (52.8%, 19/36) (p<0.0001) (Table 1). In addition, a statistically lower prevalence of anti-MCPyV antibodies, 48.1%, 26/54, was found in the group of individuals aged 5-10 years in comparison with the groups 41-50 years (64.5%, 91/141), 51-65 years (66.2%, 104/157), 71-75 years (71.7%, 33/46) (p<0.05) (Table 1).

Table 1. Seroprevalence of anti-MCPyV antibodies in healthy individuals stratified by

*p<0.0001 vs <1-4; ^Ap<0.05 vs 5-10. Bibliography

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Claims

1. An isolated peptide of Merkel cell polyomavirus (MCPyV), consisting of amino acid sequence SEQ ID NO:1 or SEQ ID NO:2.

2. An in vitro method for detecting anti- MCPyV antibodies in a biological fluid sample from a subject, comprising the steps of: a) contacting said sample with a capture agent; and b) detecting the formation of an immunocomplex consisting of the anti-MCPyV antibodies and the capture agent, wherein the formation of the immunocomplex is indicative of the presence of anti-MCPyV antibodies in the biological fluid sample, characterized in that the capture agent comprises an isolated peptide consisting of amino acid sequence SEQ ID NO:1 or an isolated peptide consisting of amino acid sequence SEQ ID NO:2 or both said isolated peptides.

3. The method according to claim 2, wherein said anti-MCPyV antibodies are human IgG and/or IgM antibodies.

4. The method according to claim 3, wherein the formation of the immunocomplex is detected by means of a secondary anti-human antibody.

5. The method according to any one of claims 2 to 4, wherein the biological fluid sample is selected from the group consisting of serum, plasma, blood, saliva, urine, or cerebrospinal fluid.

6. The method according to any one of claims 2 to 5, wherein the subject is an individual suffering from a neoplastic disease, an immunocompromised individual, an individual suffering from multiple sclerosis or from an autoimmune disease treated with a monoclonal antibody, or the subject is a healthy individual.

7. The method according to claim 6, wherein the neoplastic disease is Merkel cell carcinoma (MCC).

8. The method according to any one of claims 2 to 7, wherein the immunocomplex is detected by immunoprecipitation; immunoassay, preferably ELISA or RIA or fluorescent immunoassay; western blot; FACS analysis; or an immunocytochemistry or immunohistochemistry technique.

9. The method according to claim 8, which is an indirect ELISA immunoassay.

10. A diagnostic kit for detecting the presence of anti-MCPyV antibodies in a biological fluid sample from a subject suspected of being infected with MCPyV, comprising:

- a capture agent comprising an isolated peptide consisting of amino acid sequence SEQ ID NO:1 or an isolated peptide consisting of amino acid sequence SEQ ID NO:2 or both said isolated peptides; and

- a reagent for detecting the formation of an immunocomplex between the anti-MCPyV antibodies and said capture agent.

11. The diagnostic kit according to claim 10, comprising a secondary anti-human antibody as the reagent for detecting the immunocomplex.

12. The diagnostic kit according to claim 10 or 11, comprising an ELISA plate as the solid support for the assay.