Serological assay for detecting vaccine-related antibodies against HPV

Abstract

The invention relates to a serological assay for detecting vaccine-related antibodies against HPV. Antigens of a first type and antigens of a second type are used in the serological assay. The antigens of the first type are reactive with respect to first antibodies contained in body fluid, said antibodies being effective against vaccinated proteins which model HPV of a first type. The antigens of the second type are reactive with respect to second antibodies contained in body fluid, said antibodies being effective against vaccinated proteins which model HPV of a second type. The invention further relates to a method for identifying HPV-vaccinated persons, to a method for distinguishing between HPV-vaccinated persons and HPV-infected persons, and to a method for monitoring the vaccination success after a vaccination against HPV.

Description

[0001] Serological test for the detection of vaccine-induced antibodies against HPV

[0002] Technical field

[0003] The present disclosure relates to a serological test for the detection of vaccine-induced antibodies against HPV.

[0004] In particular, the present disclosure relates to a method for identifying HPV-vaccinated persons. Furthermore, the present disclosure relates to a method for distinguishing between HPV-vaccinated persons and persons infected with HPV.

[0005] In particular, the present disclosure also relates to a method for monitoring the success of vaccination after vaccination against HPV.

[0006] Serological tests utilize in vitro antigen-antibody reactions. These reactions can, for example, identify the body's own antibodies in a blood sample that are specific to certain antigens and target pathogens such as viruses and bacteria. The antibodies are specifically produced by a person's immune system as an immune response to exposure to these antigens, in order to protect the body. The immune system typically mounts a similar immune response when the antigens present are not directed at a pathogen, but rather at a vaccine against that pathogen.

[0007] Serological tests are used, among other things, to detect the body's own antibodies against human papillomaviruses, abbreviated HPV. One such test is described in WO 2012 / 097788 Al, which outlines a rapid test for the qualitative and / or quantitative determination of HPV antibodies contained in body fluids. While this rapid test identifies HPV antibodies, it does not provide information on whether the identified antibodies are due to vaccination or caused by an HPV-related disease.

[0008] In practice, vaccination records are usually consulted to document possible HPV vaccinations. However, these records are often lost, and the individuals themselves cannot recall whether they were vaccinated against HPV in childhood. Furthermore, such vaccination records are unknown in many countries, meaning that no documentation of any previous HPV vaccination exists at all. Against this backdrop, there is a clear need to identify HPV antibodies resulting from vaccination. This is based on the expectation of improving serological methods for this purpose. Specifically, the aim is to enable the identification of HPV-vaccinated individuals and, in particular, to differentiate between those vaccinated against HPV and those infected with HPV.

[0009] concepts

[0010] A serological method that utilizes first-type and second-type antigens offers fundamental improvements in detection. The first-type antigens are reactive with respect to first-type antibodies present in body fluids that are effective against vaccinated proteins representing first-type HPV. The second-type antigens are reactive with respect to second-type antibodies present in body fluids that are effective against vaccinated proteins representing second-type HPV. This enables the detection of vaccine-derived antibodies against HPV.

[0011] In principle, antigens of at least one additional type can be used. In this case, for example, the antigens of at least one additional type are reactive with respect to further antibodies contained in body fluids, which are effective against vaccinated proteins that depict HPV of another type. For example, the antigens of a third type are reactive with respect to third antibodies contained in body fluids, which are effective against vaccinated proteins that depict HPV of a third type. For example, the antigens of a fourth type are reactive with respect to fourth antibodies contained in body fluids, which are effective against vaccinated proteins that depict HPV of a fourth type.

[0012] In this disclosure, the term "serological method" refers in particular to or includes a serological test, a serological testing procedure, an assay, or an immunoassay. The method is preferably an in vitro method, i.e., a method performed in vitro.

[0013] In this disclosure, the term "in vitro" means, in particular, that the process is carried out outside of a living organism, i.e., not on a person. To carry out the in vitro process, at least one sample, for example of body fluid, is taken from the person concerned. For example, a single drop (approximately 25 pL) is sufficient for carrying out the proposed process.

[0014] Suitable body fluids include blood, lymph, saliva, urine, and / or sweat. For example, the body fluid could be whole blood or a derivative of whole blood, such as serum or plasma. Ideally, the body fluid could be human serum and plasma obtained from a blood sample taken via venipuncture. The body fluid could also be capillary blood or whole blood from a person's fingertip.

[0015] In this disclosure, the term "antigen" refers to proteins of any kind that trigger an immune response in the immune system of a person exposed to them. The term "antigen" in this disclosure also includes virus-like particles (VLPs).

[0016] In this disclosure, the terms “HPV of a first type” and “HPV of a second type” refer to two distinct types of HPV. In other words, the second type is different from the first type. In this disclosure, “HPV of at least one further type” refers to at least one type of HPV that is distinct from both the first and second types. If the at least one type consists of two or more types, these types are distinct from each other.

[0017] In this disclosure, the term "vaccinated proteins" refers specifically to proteins that depict a particular type of HPV and are administered to a person during vaccination to elicit an immune response. This immune response results in the formation of endogenous antibodies specific to these proteins. Because these proteins depict a particular type of HPV, the endogenous antibodies produced are equally specific to that specific type of HPV.

[0018] Such inoculated proteins can be so-called virus-like particles (VLPs). These are virus particles that do not contain nucleic acids and therefore do not replicate in target cells. For example, virus-like particles consist of viral capsomeres or possess such capsid proteins. For example, antigens of the first type are virus-like particles. For example, antigens of the second type, or at least one other type, are virus-like particles.

[0019] The proposed method is based on the assumption that in the general population, an acute HPV-related illness is, in the vast majority of cases, caused by a single HPV type. Therefore, the individual's immune system will only produce antibodies specific to this one HPV type as an immune response to the HPV-related illness.

[0020] The proposed method is further based on the assumption that an HPV vaccine covers at least two types of HPV. For example, there are currently bivalent vaccines effective against two types of HPV, quadrivalent vaccines effective against four types of HPV, and nonavalent vaccines effective against nine types of HPV. As an immune response to the HPV vaccination, an HPV-vaccinated person will therefore produce antibodies specific to one type of HPV and antibodies specific to at least one other type of HPV. These antibodies can be detected using the proposed method by utilizing at least the antigens of the first type and the antigens of the second type. The proposed method thus enables the identification of HPV-vaccinated individuals.

[0021] The proposed method also allows for the identification of individuals infected with HPV, for example, when an HPV infection is to be detected with an HPV type that is covered by one of the antigens used—that is, the antigens of the first type, the antigens of the second type, or the antigens of the further type. If, for example, only the first type or only the second type of antibodies are detected, it can be deduced that these antibodies do not originate from a vaccination but are due to an HPV infection.

[0022] In the present disclosure, the term "normal population" is to be understood in particular as a representative cross-section of apparently healthy persons who represent the total population.

[0023] In this disclosure, the term "HPV-infected" means, in particular, that the person concerned has or has had an HPV-related disease and, as a result, that the person's immune system produces or has produced antibodies against HPV. In other words, "HPV-infected" means, in particular, that the person concerned has antibodies, especially endogenous antibodies, which are caused by an HPV-related disease. Typically, antibodies against HPV are not produced simply by the presence of an HPV infection. Rather, antibodies against HPV are only produced when the HPV enters body cells and subsequently uncontrolled cell growth occurs.

[0024] In the present disclosure, the term “HPV-vaccinated” is to be understood in particular as meaning that the person concerned has antibodies, especially endogenous antibodies, which are caused by vaccination against HPV.

[0025] In a first embodiment, the proposed method serves to identify HPV-vaccinated persons and comprises the steps of: i) providing first-type antigens and second-type antigens and labeled first antibodies and labeled second antibodies, wherein the labeled first antibodies are such that they bind or are able to bind specifically to the first-type antigens, and wherein the labeled second antibodies are such that they bind or are able to bind specifically to the second-type antigens, and wherein the labeled first antibodies and the labeled second antibodies are such that, in the presence of a reactant, they cause a measurable and / or user-perceivable reaction, or, in the case of spatial clustering, result in a measurable and / or user-perceivable change;(ii) Examination of body fluid to identify vaccine-associated first antibodies and vaccine-associated second antibodies, wherein the vaccine-associated first antibodies are effective against vaccinated proteins and represent HPV type I, and wherein the vaccine-associated second antibodies are effective against vaccinated proteins and represent HPV type II, the examination being based on a competitive approach which utilizes the antigens of type I, the labeled first antibodies, the antigens of type II, and the labeled second antibodies; (iii) Evaluation of the results of step (ii) such that an HPV-vaccinated person is identified if the vaccine-associated first antibodies and, furthermore, the vaccine-associated second antibodies are identified. In this embodiment, it can therefore be determined whether the detected antibodies against HPV are due to an HPV vaccination of the person concerned.

[0026] The embodiment can be extended to include antigens of at least one further type, such as antigens of the third type and / or antigens of the fourth type. For example, it is provided that in step i) additionally antigens of the at least one further type and labeled further antibodies are provided, wherein the labeled further antibodies are such that they can bind specifically to the antigens of the at least one further type and, in the presence of a reactant, cause a measurable and / or user-perceived reaction, or, in the case of spatial clustering, result in a measurable and / or user-perceived change.

[0027] For example, it is further envisaged that in step ii), the competitive approach uses the antigens of at least one other species and the labeled additional antibodies to identify vaccine-related additional antibodies. For example, in step iii), the results of step ii) are then evaluated such that an HPV-vaccinated person is identified if the vaccine-related first antibodies, the vaccine-related second antibodies, and, in addition, the vaccine-related additional antibodies are identified.

[0028] By expanding the scope to include antigens of at least one additional type, an HPV-vaccinated person can be identified with greater certainty. This is primarily because the expansion allows for considering more than two criteria to be met in order to determine whether a person has been vaccinated against HPV.

[0029] By expanding the scope to include antigens of at least one additional type, it is also possible or facilitated to identify the vaccine used for vaccination. As already explained above, there is currently a bivalent vaccine effective against two types of HPV, a quadrivalent vaccine effective against four types of HPV, and a nonavalent vaccine effective against nine types of HPV. All of these vaccines are effective against HPV types 16 and 18.

[0030] The quadrivalent and nonavalent HPV vaccines are also effective against various other types of HPV. The antigens of at least one additional type can be used to identify a specific HPV type against which only the nonavalent vaccine is effective. This allows for the identification of individuals vaccinated with the nonavalent vaccine. Furthermore, the antigens of at least one additional type can be used to identify two HPV types against which only the nonavalent vaccine is effective, and against one of these two types the quadrivalent vaccine is also effective. This allows for differentiation between individuals vaccinated with the bivalent, quadrivalent, and nonavalent vaccines.

[0031] In a second embodiment, the proposed method serves to distinguish between HPV-vaccinated persons and HPV-infected persons, and comprises or consists of the preceding steps i) and ii) and the following step: iii) evaluation of the results of step ii) such that an HPV-vaccinated person is identified if the vaccination-related first antibodies and, furthermore, the vaccination-related second antibodies are identified, and an HPV-infected person is identified if the vaccination-related first antibodies or the vaccination-related second antibodies are identified.

[0032] This embodiment allows for the determination of whether the detected antibodies against HPV are caused by an HPV vaccination or by an HPV infection in the affected person. This makes it possible to distinguish between HPV-vaccinated individuals and those infected with HPV.

[0033] This embodiment can also be extended to include antigens of at least one further type, such as antigens of the third type and / or antigens of the fourth type. For example, it is provided that in step i) additionally antigens of the at least one further type and labeled further antibodies are provided, wherein the labeled further antibodies are such that they can bind specifically to the antigens of the at least one further type and, in the presence of a reactant, cause a measurable and / or user-perceived reaction, or, in the case of spatial clustering, result in a measurable and / or user-perceived change.

[0034] For example, it is further envisaged that in step ii), the competitive approach uses the antigens of at least one other type and the labeled additional antibodies to identify vaccine-associated additional antibodies. For example, in step iii), the results of step ii) are then evaluated such that an HPV-vaccinated person is identified if the vaccine-associated first antibodies, the vaccine-associated second antibodies, and, in addition, the vaccine-associated additional antibodies are identified, and an HPV-associated person is identified if the vaccine-associated first antibodies, the vaccine-associated second antibodies, or the vaccine-associated additional antibodies are identified. This allows for greater certainty in distinguishing between HPV-vaccinated and HPV-associated individuals.

[0035] In a third embodiment, the proposed method serves to check the success of vaccination after vaccination against HPV and comprises or consists of the preceding steps i) and ii) and the following step: iii) Evaluation of the results of step ii) such that a success of vaccination is established if the vaccination-related first antibodies and, in addition, the vaccination-related second antibodies are identified.

[0036] This design allows for the determination of whether an HPV vaccination has been successful and whether the body's own antibodies against HPV have been formed.

[0037] This embodiment can also be extended to include antigens of at least one further type, such as antigens of the third type and / or antigens of the fourth type. For example, it is provided that in step i) additionally antigens of the at least one further type and labeled further antibodies are provided, wherein the labeled further antibodies are such that they can bind specifically to the antigens of the at least one further type and, in the presence of a reactant, cause a measurable and / or user-perceived reaction, or, in the case of spatial clustering, result in a measurable and / or user-perceived change.

[0038] For example, it is further envisaged that in step ii), the competitive approach uses the antigens of at least one further type and the labeled further antibodies to identify vaccine-related further antibodies. For example, in step iii), the results of step ii) are then evaluated such that vaccination success is established if the vaccine-related first antibodies, the vaccine-related second antibodies, and, in addition, the vaccine-related further antibodies are identified. This allows for greater certainty in monitoring vaccination success. In the proposed method, the antigens of the first type can comprise a protein complex with at least one conformational epitope, wherein this protein complex contains or is composed of aggregated proteins representing vaccinated proteins that map the Ll protein of HPV of the first type.Alternatively, the antigens of the first type may comprise an amino acid sequence, in particular a polypeptide, with at least one linear epitope, wherein this amino acid sequence represents inoculated proteins that map the Ll protein of HPV of the first type.

[0039] In the proposed method, the second-type antigens can comprise a protein complex with at least one conformational epitope, wherein this protein complex includes or is composed of aggregated proteins representing inoculated proteins that mimic the Ll protein of the second-type HPV. Alternatively, the second-type antigens can comprise an amino acid sequence, in particular a polypeptide, with at least one linear epitope, wherein this amino acid sequence represents inoculated proteins that mimic the Ll protein of the second-type HPV.

[0040] In the proposed method, the antigens of at least one further type can comprise a protein complex with at least one conformational epitope, wherein this protein complex includes or is composed of aggregated proteins representing inoculated proteins that mimic the Ll protein of HPV of the further type. Alternatively, the antigens of at least one further type can comprise an amino acid sequence, in particular a polypeptide, with at least one linear epitope, wherein this amino acid sequence represents inoculated proteins that mimic the Ll protein of HPV of the further type.

[0041] A serological method using antigens comprising the protein complex with at least one conformational epitope is described in more detail in publication WO 2012 / 097788 Al, to which reference is hereby made for the purpose of completing and supplementing the present disclosure, with the note that the publication may assign a meaning to identical terms which differs from the meaning given here.

[0042] A serological method using antigens comprising an amino acid sequence with at least one linear epitope is described in more detail in the unpublished German patent application DE 10 2022 127 779.8 and international patent application PCT / DE2023 / 100785, to which reference is hereby made for the purpose of completing and supplementing the present disclosure, with the note that the patent applications may assign meanings to identical terms that differ from the meaning used here. The amino acid sequence there is such a sequence or linear epitope that occurs in all high-risk HPV types. In the present case, the amino acid sequence or linear epitope is chosen to be specific for a single HPV type, for example, HPV type 1, HPV type 2, or HPV of at least one other type.

[0043] The proposed method can be designed such that the first-type antigens are reactive against antibodies that are effective against HPV type 16 and / or against vaccinated proteins that reflect HPV type 16. In particular, the first antibodies are effective against HPV type 16 and / or against vaccinated proteins that reflect HPV type 16. Specifically, the labeled first antibodies are also effective against HPV type 16 and / or against vaccinated proteins that reflect HPV type 16.

[0044] The proposed method can further be designed such that the second-type antigens are reactive against antibodies that are effective against HPV type 18 and / or against vaccinated proteins that reflect HPV type 18. In particular, the second-type antibodies are effective against HPV type 18 and / or against vaccinated proteins that reflect HPV type 18. Specifically, the labeled second-type antibodies are also effective against HPV type 18 and / or against vaccinated proteins that reflect HPV type 18.

[0045] It has been shown that antibodies specific to HPV type 16 are very rare in the unvaccinated general population. Studies have observed that approximately 1 to 2 people per 1000 have such antibodies against HPV type 16. Furthermore, it has been shown that antibodies specific to HPV type 18 are even rarer in the unvaccinated general population.

[0046] Studies have shown that approximately 1 in 1000 people have antibodies against HPV type 18. Statistically, this suggests that in the unvaccinated general population, approximately 1 in 1 million people should have antibodies against both HPV type 16 and HPV type 18. Furthermore, all current HPV vaccines cover at least HPV types 16 and 18, which are primarily responsible for the development of carcinomas. Therefore, the proposed serological test, with its combined detection of antibodies against at least two HPV types, particularly facilitates the identification of a vaccinated individual when focusing on antibodies against both HPV type 16 and HPV type 18.

[0047] Alternatively, the proposed method can be designed such that the antigens of the first type are reactive against antibodies that are effective against HPV type 6 and / or against vaccinated proteins that reflect HPV type 6. In particular, the first antibodies are effective against HPV type 6 and / or against vaccinated proteins that reflect HPV type 6. Specifically, the labeled first antibodies are also effective against HPV type 6 and / or against vaccinated proteins that reflect HPV type 6. In principle, the antigens of the second type can also be reactive against antibodies that are effective against HPV type 6 and / or against vaccinated proteins that reflect HPV type 6.

[0048] Alternatively, the proposed method can be designed such that the second-type antigens are reactive against antibodies effective against HPV type 11 and / or against vaccinated proteins that reflect HPV type 11. In particular, the second-type antibodies are effective against HPV type 11 and / or against vaccinated proteins that reflect HPV type 11. Specifically, the labeled second-type antibodies are also effective against HPV type 11 and / or against vaccinated proteins that reflect HPV type 11. In principle, the first-type antigens can also be reactive against antibodies effective against HPV type 11 and / or against vaccinated proteins that reflect HPV type 11.

[0049] HPV types 6 and 11 are primarily responsible for the development of genital warts. A proposed serological test, which combines the detection of antibodies against at least two HPV types, can identify individuals vaccinated against genital warts, for example, by focusing on antibodies against HPV type 6 and HPV type 11.

[0050] In principle, it can also be provided that the antigens of the first type are reactive against antibodies effective against vaccinated proteins that reflect HPV of one of the types 6, 11, 16, 18, 31, 33, 45, 52, or 58, and that the antigens of the second type are reactive against antibodies effective against vaccinated proteins that reflect HPV of another of the types 6, 11, 16, 18, 31, 33, 45, 52, or 58. Additionally, it can be provided that the antigens of at least one further type are reactive against antibodies effective against vaccinated proteins that reflect HPV of yet another of the types 6, 11, 16, 18, 31, 33, 45, 52, or 58. This is based on the fact that the nine-component vaccine described above is effective against these nine types 6, 11, 16, 18, 31, 33, 45, 52, 58 of HPV, the four-component vaccine described above is effective against types 6, 11, 16, 18, and the two-component vaccine described above is effective against types 16, 18.This means that the serological test can also determine which of the three vaccines a person vaccinated against HPV has received.

[0051] Such identification is possible if reference is made to HPV types 16 and 18, furthermore to at least one of HPV types 6 and 11, and additionally to at least one of HPV types 31, 33, 45, 52, or 58. One possible implementation is that the antigens of the first type are reactive against antibodies effective against vaccinated proteins that reflect HPV type 16; that the antigens of the second type are reactive against antibodies effective against vaccinated proteins that reflect HPV type 18; that the antigens of a third type are reactive against antibodies effective against vaccinated proteins that reflect HPV type 6 or 11; and that the antigens of a fourth type are reactive against antibodies effective against vaccinated proteins that reflect HPV type 31, 33, 45, 52, or 58.

[0052] Example 1

[0053] One possible embodiment of a serological test for the detection of antibodies against HPV uses antigens of a first type and antigens of a second type. The antigens of the first type are reactive with respect to first antibodies contained in body fluid, which are effective against vaccinated proteins that reflect HPV type 16 (HPV16). The antigens of the second type are reactive with respect to second antibodies contained in body fluid, which are effective against vaccinated proteins that reflect HPV type 18 (HPV18).

[0054] For example, the first type of antigen comprises a protein complex with conformational epitopes on its surface, which are designed to bind the first type of antibodies specifically. For example, a protein complex with aggregated proteins is used that presents inoculated proteins that mimic the Ll protein of HPV16. Similarly, the second type of antigen comprises a protein complex with conformational epitopes on its surface, which are designed to bind the second type of antibodies specifically. For example, a protein complex with aggregated proteins is used that presents inoculated proteins that mimic the Ll protein of HPV18.

[0055] For example, the antigens of the first type are stored in a liquid, in particular a storage liquid, which is physiologically active on the antigens of the first type in order to maintain the reactivity of the antigens of the first type with respect to the first antibodies. For example, the antigens of the second type are stored in a liquid, in particular a storage liquid, which is physiologically active on the antigens of the second type in order to maintain the reactivity of the antigens of the second type with respect to the second antibodies.

[0056] For example, the liquid for the first-type antigens and the second-type antigens is the same liquid or a similar liquid. The first-type and second-type antigens can be stored together in one reagent containing the liquid. Alternatively, the first-type antigens can be stored in a first reagent and the second-type antigens in a second reagent, each containing the liquid. The liquid or storage liquid preferably has a pH between approximately 7.0 and approximately 9.0. The liquid or storage liquid can be a saline solution or TRIS-BSA.

[0057] For example, in the serological test, labeled first antibodies and labeled second antibodies are used to identify the first and second antibodies. The labeled first antibodies have the property of specifically binding the antigens of the first type, and the labeled second antibodies have the property of specifically binding the antigens of the second type.

[0058] For example, the labeled first antibodies are designed to bind specifically to the configurational epitopes of the first-type antigens described above. For example, the labeled second antibodies are also designed to bind specifically to the configurational epitopes of the second-type antigens described above. For example, the labeled first antibodies and the labeled second antibodies are obtained from a single cell line, in particular from a hybridoma cell line. For example, the labeled first antibodies and / or the labeled second antibodies are labeled with gold particles.

[0059] Performing a serological test involves first combining bodily fluids containing type I and type II antigens. These bodily fluids could be, for example, human serum and plasma from a blood sample. The blood sample can be obtained through venipuncture or from a finger prick. The blood sample can be a single sample for type I and type II antigens, or it can be a separate sample for type I and type II antigens.

[0060] The serological test further involves, in a second step, preferably performed at a later time, the combination of labeled first antibodies with the body fluid and the first-type antigens, and the combination of labeled second antibodies with the body fluid and the second-type antigens. The labeled first antibodies are combined with the body fluid and the first-type antigens to elicit a measurable and / or user-perceived reaction, or, in the case of spatial clustering, a measurable and / or user-perceived change, when the labeled first antibodies bind to the first-type antigens.The labeled second antibodies are brought together with the body fluid and the second type antigens to produce a measurable and / or user-perceived response or, in the case of spatial clustering, a measurable and / or user-perceived change when the labeled second antibodies bind to the second type antigens.

[0061] The serological test is based on the premise that the labeled first antibodies bind specifically to the first-type antigens, resulting in a measurable and / or user-perceived reaction or change, provided the aforementioned first antibodies are not present in the body fluid. However, if the aforementioned first antibodies are present in the body fluid, these first antibodies bind specifically to the first-type antigens as soon as the body fluid is brought into contact with the first-type antigens. The specific binding sites of the first-type antigens are then already occupied or largely occupied when the second step is carried out, in which the labeled first antibodies are brought into contact with the body fluid and the first-type antigens.A specific binding of the labeled first antibodies to the antigens of the first type, and thus a measurable and / or user-perceived reaction or change, then does not occur or largely does not occur.

[0062] The serological test is further based on the premise that the labeled second-type antibodies also bind specifically to the second-type antigens, resulting in a measurable and / or user-perceived reaction or change, provided the aforementioned second-type antibodies are not present in the body fluid. However, if the aforementioned second-type antibodies are present in the body fluid, these antibodies bind specifically to the second-type antigens as soon as the body fluid is brought into contact with the second-type antigens. The specific binding sites of the second-type antigens are then already occupied or largely occupied when the second step is carried out, in which the labeled second-type antibodies are brought into contact with the body fluid and the second-type antigens.A specific binding of the labeled second type antibodies to the antigens of the second type, and thus a measurable and / or user-perceived reaction or change, then does not occur or largely does not occur.

[0063] For example, the labeled first antibodies are mobile on a carrier medium. The carrier medium can be a test strip. For example, the carrier medium has a porous membrane which, for example, due to capillary action, promotes the flow of a liquid in at least one spatial axis. For example, the carrier medium contains first capture molecules. For example, the first capture molecules are fixed in a predefined zone of the carrier medium, for example, spatially spaced from the labeled first antibodies.

[0064] For example, the first capture molecules have the property of specifically binding the first-type antigens in order to cause a measurable and / or user-perceived change in the area containing the first capture molecules when the first capture molecules bind to the first-type antigens. For example, the first capture molecules are designed to specifically bind to the conformational epitopes of the first-type antigens described above. For example, the first capture molecules are obtained from a hybridoma cell line. For example, the first capture molecules are antibodies corresponding to the labeled first antibodies, except that, unlike the labeled first antibodies, the first capture molecules are not labeled.

[0065] For example, the labeled first antibodies are present in a predetermined quantity. This predetermined quantity is such that, upon reaching the zone containing the first capture molecules, the first-type antigens still have free binding sites available for binding to these molecules. If the body fluid containing the first-type antigens is then applied to the carrier medium, for example, into an application zone, a flow movement towards the first capture molecules occurs due to the liquid phase and capillary action.

[0066] If the first antibodies described above are not present in the body fluid, the labeled first antibodies bind specifically to the first-type antigens as described above. This causes the labeled first antibodies to flow along with the first-type antigens and the body fluid, and upon reaching the zone, they come into contact with the first capture molecules present there. The first capture molecules will bind specifically to any remaining free binding sites on the first-type antigens, causing the labeled first antibodies to accumulate in this zone and resulting in a measurable and / or user-perceived change.

[0067] For example, the labeled second antibodies are mobile on a further carrier medium. This further carrier medium can also be a test strip. For example, the further carrier medium has a porous membrane which, for example, due to capillary action, promotes the flow of a liquid in at least one spatial axis. For example, the further carrier medium contains second capture molecules. For example, the second capture molecules are fixed in a predefined zone of the further carrier medium, for example, spatially spaced from the labeled second antibodies.

[0068] For example, the second capture molecules have the property of specifically binding the second-type antigens in order to cause a measurable and / or user-perceived change in the area containing the second capture molecules when the second capture molecules bind to the second-type antigens. For example, the second capture molecules are designed to specifically bind to the configurational epitopes of the second-type antigens described above. For example, the second capture molecules are derived from a hybridoma cell line. For example, the second capture molecules are antibodies corresponding to the labeled second antibodies, except that, unlike the labeled second antibodies, the second capture molecules are not labeled.

[0069] For example, the labeled second antibodies are present in a predetermined quantity. Preferably, the predetermined quantity of the labeled second antibodies is such that, upon reaching the zone containing the second capture molecules, the first-type antigens still have free binding sites for the second capture molecules. If the body fluid containing the second-type antigens is then applied to the carrier medium, for example, to an application zone, a flow movement towards the second capture molecules occurs due to the liquid phase and capillary action.

[0070] If the second type of antibodies described above are not present in the body fluid, the labeled second type of antibodies bind specifically to the second type of antigens as described above. This causes the labeled second type of antibodies to migrate along with the second type of antigens and the body fluid, and upon reaching the zone, they come into contact with the second type of capture molecules present there. The second type of capture molecules will bind specifically to any remaining free binding sites on the second type of antigens, causing the labeled second type of antibodies to accumulate in this zone and resulting in a measurable and / or user-perceived change.

[0071] The serological test makes it possible to identify HPV-vaccinated individuals. The serological test is based on the premise that, in the general population, an acute HPV-related illness is in the vast majority of cases caused by a single HPV type. Therefore, the individual's immune system will only produce antibodies specific to that one HPV type as an immune response to the HPV-related illness.

[0072] The serological test is further based on the assumption that an HPV vaccine covers at least two types of HPV. For example, a bivalent vaccine effective against two types of HPV, a quadrivalent vaccine effective against four types of HPV, and a nonavalent vaccine effective against nine types of HPV are currently in use. All of these vaccines are directed at least against HPV types 16 and 18, i.e., against HPV16 and HPV18. Therefore, the serological test preferably focuses on antibodies against HPV type 16 and antibodies against HPV type 18.

[0073] The performance of the serological test for identifying HPV-vaccinated individuals is described below using the example of an examination of samples from 20 different individuals. The samples were serum samples, specifically whole blood samples, from these 20 different individuals. Each sample was examined using the serological test for the presence of endogenous antibodies against HPV16 and the presence of endogenous antibodies against HPV18, following the procedure described above.

[0074] The results of the serological tests are summarized in Table 1. The individual samples are listed under the heading "Sample" with values ​​from "Sl" to "S20". The result of the test for autoantibodies against HPV16 is listed under "HPV16AK". The result of the test for autoantibodies against HPV18 is listed under "HPV18AK". The result is marked "positive" if the serological test detected antibodies against HPV16, i.e., the first antibodies. The result is marked "negative" if the serological test did not detect antibodies against HPV18, i.e., the second antibodies.

[0075] For comparison with the examination results, the heading "Status" indicates whether the respective person has actually been vaccinated against HPV ("vaccinated"). This information was obtained by interviewing the person, by reviewing their vaccination record, or through a reference examination. Information on whether the person had or has HPV16 and / or HPV18 infection was also obtained by interviewing the person or through a reference examination. In this case, Table 1 indicates "HPV16 infection" or "HPV18 infection." If no evidence of HPV vaccination or HPV infection was found or detected in a reference examination, these individuals are listed as "healthy" in Table 1. Table 1

[0076] As can be seen in Table 1, an individual is identified as HPV-vaccinated if both the first antibodies ("HPV16AK") and the second antibodies ("HPV18AK") are detected in the sample. This result corresponds in all cases with the information under "Status".

[0077] As can be seen from Table 1, the serological test is also suitable for differentiating between HPV-vaccinated individuals and those infected with HPV. This is the case when either the first antibodies ("HPV16AK") or the second antibodies ("HPV18AK") are detected in the sample. This result also corresponds in all cases with the information under "Status". Furthermore, the serological test is also suitable for determining whether an HPV-infected individual has an HPV16-related disease or an HPV18-related disease. An HPV16-related disease is identified if the first antibodies ("HPV16AK") are detected, but the second antibodies ("HPV18AK") are not. Conversely, an HPV18-related disease is identified if the first antibodies ("HPV16AK") are not detected, but the second antibodies ("HPV18AK") are detected.

[0078] As can be seen from Table 1, a negative result regarding the first antibodies (“HPV16AK”) and a negative result regarding the second antibodies (“HPV18AK”) can indicate that the person concerned has neither been vaccinated against HPV nor has or had an HPV-related disease.

[0079] Example 2

[0080] Another possible embodiment of a serological test for the detection of antibodies against HPV uses antigens of a first type, antigens of a second type, antigens of a third type, and antigens of a fourth type. This embodiment differs from the previous embodiment (Example 1) of the serological test in that, in addition to the antigens of the first type and the antigens of the second type, the antigens of the third type and the antigens of the fourth type are also provided.

[0081] Regarding the antigens of the first type and the antigens of the second type, reference is made to the description of the preceding embodiment. The antigens of the third type are, for example, those antigens that are reactive with respect to third-type antibodies contained in body fluids, which are effective against vaccinated proteins that express HPV type 6 (HPV6). Alternatively, the antigens of the third type can also be those antigens that are reactive with respect to third-type antibodies contained in body fluids, which are effective against vaccinated proteins that express HPV type 11 (HPV11).

[0082] Fourth-type antigens are, for example, those antigens that are reactive with respect to fourth-type antibodies present in body fluids, which are effective against proteins expressed by HPV type 31 (HPV31). Alternatively, fourth-type antigens can also be those antigens that are reactive with respect to fourth-type antibodies present in body fluids, which are effective against proteins expressed by HPV type 33 (HPV33). Alternatively, fourth-type antigens can also be those antigens that are reactive with respect to fourth-type antibodies present in body fluids, which are effective against proteins expressed by HPV type 45 (HPV45). Alternatively, fourth-type antigens can also be those antigens that are reactive with respect to fourth-type antibodies present in body fluids, which are effective against proteins expressed by HPV type 52 (HPV52).Alternatively, the fourth type antigens can also be antigens that are reactive with respect to fourth antibodies contained in body fluid, which are effective against vaccinated proteins that depict HPV type 58 (HPV58).

[0083] For example, the structure and function of the serological test are identical with regard to the additional HPV types considered compared to the structure and function of the serological test with regard to the already considered HPV types 16 and 18. HPV types 6 and 31 are considered below as examples of additional HPV types.

[0084] For example, third-type antigens comprise a protein complex with conformational epitopes on their surface, which are designed to specifically bind third-type antibodies. For instance, a protein complex with aggregated proteins is used that presents inoculated proteins mimicking the Ll protein of HPV6.

[0085] For example, fourth-type antigens comprise a protein complex with conformational epitopes on their surface, which are designed to specifically bind the fourth-type antibodies. For instance, a protein complex with aggregated proteins is used that presents inoculated proteins mimicking the Ll protein of HPV31.

[0086] For example, the third-type antigens are stored in the liquid or storage fluid described above, which has a physiological effect on the third-type antigens in order to maintain their reactivity with respect to the third-type antibodies. For example, the fourth-type antigens are also stored in such a liquid.

[0087] For example, in the serological test, labeled third antibodies and labeled fourth antibodies are used to identify the third and fourth antibodies. The labeled third antibodies have the property of specifically binding to third-type antigens, and the labeled fourth antibodies also have the property of specifically binding to fourth-type antigens.

[0088] For example, the labeled third-type antibodies are mobile and mounted on a carrier medium. The carrier medium may contain third-type capture molecules. Alternatively, the third-type capture molecules may be fixed within a predefined zone of the carrier medium, for example, spatially spaced from the labeled third-type antibodies. The third-type capture molecules may have the property of specifically binding the third-type antigens to cause a measurable and / or user-perceived change in the area containing the third-type capture molecules when the third-type capture molecules bind to the third-type antigens.

[0089] For example, the labeled fourth-type antibodies are mobilized on a further carrier medium. Preferably, this further carrier medium contains fourth-type capture molecules. For example, the fourth-type capture molecules are fixedly arranged in a predefined zone of the further carrier medium, for example, spatially spaced apart from the labeled fourth-type antibodies. For example, the fourth-type capture molecules have the property of specifically binding the fourth-type antigens in order to cause a measurable and / or user-perceived change in the region containing the fourth-type capture molecules when the fourth-type capture molecules bind to the fourth-type antigens.

[0090] In this exemplary embodiment of the serological test, it is possible to determine the type of vaccine used. It can differentiate whether a vaccinated person received the bivalent, quadrivalent, or nonavalent vaccine. This is because, unlike the bivalent vaccine, the quadrivalent and nonavalent vaccines are effective against HPV6, and only the nonavalent vaccine is also effective against HPV31.

[0091] To demonstrate the performance of this embodiment, the samples from the 20 different individuals already described in the previous embodiment (Example 1) are used below and additionally examined using the serological test for the presence of endogenous antibodies against HPV6 and the presence of endogenous antibodies against HPV31.

[0092] The results of the serological tests are summarized in Table 2.

[0093] Table 2 Table 2 differs from Table 1 in that the result of the test for autoantibodies against HPV6 is listed under the designation "HPV6AK," and the result of the test for autoantibodies against HPV31 is listed under the designation "HPV31AK." A result is marked "positive" if the serological test detects antibodies against HPV6, i.e., the third antibody. A result is marked "negative" if the serological test detects antibodies against HPV31, i.e., the fourth antibody.

[0094] For comparison with the examination results, the heading "Status" indicates whether the individual has actually been vaccinated against HPV and, if so, whether the vaccination was administered with the bivalent vaccine ("12"), the quadrivalent vaccine ("14"), or the nonavalent vaccine ("19"). This information was obtained by interviewing the individual, by reviewing their vaccination record, or through a reference examination.

[0095] As can be seen from Table 2, an individual is identified as HPV-vaccinated if both the first antibodies (“HPV16AK”) and the second antibodies (“HPV18AK”) are detected in the sample. For these HPV-vaccinated individuals, the bivalent vaccine is identified as the vaccine used if neither the third antibodies (“HPV6AK”) nor the fourth antibodies (“HPV31AK”) are detected in the sample (see samples “S10” and “S19”).

[0096] Furthermore, the quadrivalent vaccine is identified as the vaccine used if the third antibody ("HPV6AK") but no fourth antibody ("HPV31AK") was detected in the sample tested (see sample "S13"). Additionally, the nine-valent vaccine is identified as the vaccine used if both the third antibody ("HPV6AK") and the fourth antibody ("HPV31AK") were detected in the sample tested (see sample "S4").

[0097] The boundary values ​​for the ranges specified in the present disclosure also include, in particular, the boundary values ​​themselves for the respective range. Furthermore, any individual values ​​contained within the specified ranges are also included. Moreover, as those skilled in the art know, the lower and upper limits of the range specifications mentioned in the present disclosure are freely combinable.

Claims

Patent claims 1. Serological method for the detection of vaccine-induced antibodies against HPV, in which antigens of a first type and antigens of a second type are used, wherein the antigens of the first type are reactive with respect to first antibodies contained in body fluid which are effective against vaccinated proteins that represent HPV of a first type, wherein the antigens of the second type are reactive with respect to second antibodies contained in body fluid which are effective against vaccinated proteins that represent HPV of a second type.

2. Serological method according to claim 1, wherein antigens of at least one further type are used, the antigens of the at least one further type being reactive with respect to further antibodies contained in body fluid which are effective against vaccinated proteins that represent HPV of a further type.

3. Serological method for identifying HPV-vaccinated individuals, comprising or consisting of the steps of: i) providing first-type antigens and second-type antigens and labeled first antibodies and labeled second antibodies, wherein the first-type antigens are reactive with respect to first antibodies contained in body fluid that are effective against vaccinated proteins representing first-type HPV, and the second-type antigens are reactive with respect to second antibodies contained in body fluid that are effective against vaccinated proteins representing second-type HPV, wherein the labeled first antibodies are designed to bind specifically to the first-type antigens, and the labeled second antibodies are designed to bind specifically to the second-type antigens.wherein the labeled first antibodies and the labeled second antibodies are such that, in the presence of a reactant, they produce a measurable and / or user-perceived response, or, in spatial clustering, produce a measurable and / or user-perceived change; ii) examination of body fluid to identify vaccine-derived first antibodies and vaccine-derived second antibodies, wherein the vaccine-derived first antibodies are effective against vaccinated proteins and reflect type 1 HPV, and which, vaccine-induced second antibodies are effective against vaccinated proteins and reflect the second type of HPV, using a competitive approach that utilizes the first type antigens, the labeled first antibodies, the second type antigens, and the labeled second antibodies; iii) evaluation of the results of step ii) to the effect that an HPV-vaccinated person is identified if the vaccine-induced first antibodies and, in addition, the vaccine-induced second antibodies are identified.

4. Serological method according to claim 3, wherein in step i) antigens of at least one further type and labeled further antibodies are additionally provided, wherein the antigens of the at least one further type are reactive with respect to further antibodies contained in body fluid which are effective against vaccinated proteins and which represent HPV of a further type, wherein the labeled further antibodies are such that they can bind specifically to the antigens of the at least one further type and, in the presence of a reactant, cause a measurable and / or user-perceived reaction or, in the case of spatial clustering, result in a measurable and / or user-perceived change, wherein in step ii) the competitive approach uses the antigens of the at least one further type and the labeled further antibodies to identify vaccine-induced further antibodies.and in step iii) the results of step ii) are evaluated to determine that an HPV-vaccinated person is identified if the vaccine-related first antibodies, the vaccine-related second antibodies, and, in addition, the vaccine-related further antibodies are identified.

5. Serological method for differentiating between HPV-vaccinated persons and HPV-infected persons, comprising or consisting of the steps of: i) providing first-type antigens and second-type antigens and labeled first antibodies and labeled second antibodies, wherein the first-type antigens are distinguished with respect to the first antibodies contained in body fluid are reactive, which are effective against vaccinated proteins that depict HPV of a first type, and wherein the antigens of the second type are reactive with respect to second antibodies contained in body fluid Antibodies are reactive which are effective against vaccinated proteins that depict HPV of a second type, wherein the labeled first antibodies are such that they can bind specifically to the antigens of the first type, and wherein the labeled second antibodies are such that they can bind specifically to the antigens of the second type, wherein the labeled first antibodies and the labeled second antibodies are such that, in the presence of a reactant, they cause a measurable and / or user-perceived reaction or, in spatial clustering, result in a measurable and / or user-perceived change;(ii) Examination of body fluid to identify vaccine-associated first antibodies and vaccine-associated second antibodies, wherein the vaccine-associated first antibodies are effective against vaccinated proteins and reflect HPV type 1, and the vaccine-associated second antibodies are effective against vaccinated proteins and reflect HPV type 2, the examination being based on a competitive approach which utilizes the antigens of type 1, the labeled first antibodies, the antigens of type 2, and the labeled second antibodies; (iii) Evaluation of the results of step (ii) such that an HPV-vaccinated person is identified if the vaccine-associated first antibodies and, furthermore, the vaccine-associated second antibodies are identified, and an HPV-infected person is identified if either the vaccine-associated first antibodies or the vaccine-associated second antibodies are identified.

6. Serological method according to claim 5, wherein in step i) additionally antigens of at least one further type and labeled further antibodies are provided, wherein the antigens of the at least one further type are reactive with respect to further antibodies contained in body fluid which are effective against vaccinated proteins and which represent HPV of a further type, wherein the labeled further antibodies are such that they can bind specifically to the antigens of the at least one further type and, in the presence of a reactant, cause a measurable and / or user-perceived reaction or, in the case of spatial clustering, result in a measurable and / or user-perceived change. In step ii) the competitive approach uses the antigens of at least one other type and the labeled further antibodies to identify vaccine-related further antibodies, and in step iii) the results of step ii) are evaluated to determine that an HPV-vaccinated person is identified if the vaccine-related first antibodies and the vaccine-related second antibodies and, in addition, the vaccine-related further antibodies are identified, and that an HPV-infected person is identified if the vaccine-related first antibodies or the vaccine-related second antibodies or the vaccine-related further antibodies are identified.

7. Serological method for monitoring the success of vaccination after vaccination against HPV, comprising or consisting of the steps of: i) providing first-type antigens and second-type antigens and labeled first antibodies and labeled second antibodies, wherein the first-type antigens are reactive with respect to first antibodies contained in body fluids which are effective against vaccinated proteins representing first-type HPV, and wherein the second-type antigens are reactive with respect to second antibodies contained in body fluids which are effective against vaccinated proteins representing second-type HPV, wherein the labeled first antibodies are such that they can bind specifically to the first-type antigens, and wherein the labeled second antibodies are such that they can bind specifically to the second-type antigens.wherein the labeled first antibodies and the labeled second antibodies are such that, in the presence of a reactant, they produce a measurable and / or user-perceived response, or, in spatial clustering, produce a measurable and / or user-perceived change; ii) Examination of body fluid to identify vaccine-derived first antibodies and vaccine-derived second antibodies, wherein the vaccine-derived first antibodies are effective against vaccinated proteins and reflect type I HPV, and the vaccine-derived second antibodies are effective against vaccinated proteins and reflect type II HPV, wherein the examination is based on a competitive approach which identifies the type I antigens, the labeled first, Antibodies that utilize second-type antigens and the labeled second antibodies; iii) evaluation of the results of step ii) to the effect that a vaccination success is established if the vaccination-induced first antibodies and, in addition, the vaccination-induced second antibodies are identified.

8. Serological method according to claim 7, wherein in step i) antigens of at least one further type and labeled further antibodies are additionally provided, wherein the antigens of the at least one further type are reactive with respect to further antibodies contained in body fluid which are effective against vaccinated proteins and which represent HPV of a further type, wherein the labeled further antibodies are such that they can bind specifically to the antigens of the at least one further type and, in the presence of a reactant, cause a measurable and / or user-perceived reaction or, in the case of spatial clustering, result in a measurable and / or user-perceived change, wherein in step ii) the competitive approach uses the antigens of the at least one further type and the labeled further antibodies to identify vaccine-induced further antibodies,and in step iii) the results of step ii) are evaluated to such an extent that a vaccination success is established if the vaccination-related first antibodies and the vaccination-related second antibodies and, in addition, the vaccination-related further antibodies are identified.

9. Serological method according to one of the preceding claims, wherein the antigens of the first type comprise a protein complex with at least one conformational epitope and this protein complex has or is formed from aggregated proteins which represent inoculated proteins that map the Ll protein of HPV of the first type, or comprise an amino acid sequence, in particular a polypeptide, with at least one linear epitope and this amino acid sequence represents inoculated proteins that map the Ll protein of HPV of the first type, and wherein the antigens of the second type comprise a protein complex with at least one conformational epitope and this protein complex has or is formed from aggregated proteins which represent inoculated proteins represent the Ll protein of type 2 HPV, or comprise an amino acid sequence, in particular a polypeptide, with at least one linear epitope, and this amino acid sequence represents inoculated proteins that represent the Ll protein of type 2 HPV.

10. Serological method according to one of claims 2, 4, 6, 8, wherein the antigens of at least one further type comprise a protein complex with at least one conformational epitope and this protein complex has aggregated proteins or is formed from which represent inoculated proteins that depict the Ll protein of HPV of one further type, or comprise an amino acid sequence, in particular a polypeptide, with at least one linear epitope and this amino acid sequence represents inoculated proteins that depict the Ll protein of HPV of one further type.

11. Serological method according to one of the preceding claims, wherein the antigens of the first type are reactive against antibodies effective against vaccinated proteins that reflect HPV type 16, and wherein the antigens of the second type are reactive against antibodies effective against vaccinated proteins that reflect HPV type 18.

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