Environmental population immune response

Abstract

A method for measuring and generating an alert of vaccination response of an epidemiological unit producing an animal, the method comprising determining and evaluating an environmental population immune response from an air sample.

Description

Technical Field

[0001] The present invention relates to the field of animal health and uses a variety of molecular and epidemiological techniques to determine and assess the immune response of environmental populations to vaccines in the environment of epidemiological units of production animals. Background Art

[0002] In livestock farming settings, proper monitoring and management of animal health and welfare are key aspects of ensuring the production of safe, high-quality food, as well as disease prevention and control, especially in confined environments where pathogens can spread rapidly and the risk of infection is high (US2022026318 A1). In the field of zooepidemiology or veterinary epidemiology, monitoring and analysis of animal populations are crucial for understanding disease dynamics, which helps implement appropriate management strategies and evaluate the effectiveness of interventions such as vaccination and treatment (Pardo MV, 2006).

[0003] In this context, the effectiveness of vaccination strategies in animal health is a fundamental and central factor in evaluating interventions and treatments. This makes ongoing animal welfare surveillance crucial, particularly focused on assessing immune responses following vaccination. This approach allows us to determine how animals respond to introduced immunizations and their effectiveness in providing protection against environmental pathogens. Therefore, the ability to monitor and evaluate vaccine effectiveness is crucial. This not only facilitates early detection of vaccine effectiveness but also allows for rapid and appropriate intervention to strengthen herd immunity and reduce disease transmission within animal populations.

[0004] Traditionally, biomarker analysis and serological methods have been used to assess immune responses in animals. However, these methods are costly, time-consuming, and labor-intensive, and they are limited in the types of pathogens they assess. Furthermore, they do not fully reflect the immune responses of animal populations.

[0005] Prior art methods, such as analysis of biomarkers such as inflammatory proteins or cytokines, have been used to assess immune responses in animals (Marcelo Ocamo, 2010). Other methods, such as ELISA, SN, or IPMA, offer high precision and characterize the immune response by detecting antibody activity in plasma or tissue samples from animals (Immunology Research Unit, 2018). Specifically, ELISA has been used to detect a variety of target analytes in different sample types. However, these methods can be costly, labor-intensive, and limited in the types of pathogens they assess (Hosseini et al., 2018). Furthermore, due to biases in the selection of sampled animals and variations in the quality and quantity of the samples obtained, these methods may not accurately reflect the immune response of the entire animal population being assessed, particularly during the early stages of infection when prevalence is low within the population.

[0006] On the other hand, the understanding of the immune response to specific pathogens (such as mycoplasmas) is limited when using only serological methods, and more advanced and less readily available methods, such as monitoring cellular responses, are needed to fully understand these pathogens. In this context, monitoring vaccine shedding becomes an important strategy to infer the level of protection in advance.

[0007] Meanwhile, the fight against highly prevalent pathogens in agricultural settings isn't solely focused on boosting immune responses through vaccines; it also seeks to establish interference between attenuated vaccine strains and endemic viruses. This interference between vaccine and endemic strains occurs through competition for cellular receptor sites, allowing the vaccine strain to displace the pathogenic strain at high concentrations. Environmental monitoring of vaccine virus levels can reveal the extent of protection conferred by vaccine strain interference, representing a significant advance in managing and understanding immunity in animal populations.

[0008] Recent studies have demonstrated non-invasive methods for diagnosing disease using biomarkers in exhaled lung fluid droplets, opening the possibility of applying similar techniques to monitor immune responses in animals (Morozov et al., 2018). Furthermore, studies have shown that air-dried saliva and fecal samples can be used for semi-quantitative measurement of mucosal antibodies, which could simplify the collection and analysis of immunological data in agricultural settings (Vetvik et al., 1998). Meanwhile, the use of environmental eDNA / eRNA for pathogen detection and monitoring offers a promising approach for non-invasive animal health monitoring (Bass et al., 2023).

[0009] In the patent landscape, some techniques have been explored to determine the immune response of birds to attenuated vaccines, although these methods still require invasive procedures (CZ309268B6). Other studies have focused on sequence-based measures to assess immune responses, supporting the use of molecular biology for such purposes (US2014356339 AA). Furthermore, studies have described methods for predicting immune responses at the population level, demonstrating growing interest in collective immunity assessment (US2008220450AA).

[0010] This prospect highlights the need and potential for improved methods that can accurately and non-invasively assess animal immune responses, which are critical for effective health management of production animal populations.

[0011] The present invention proposes a method to inform the level of animal protection in a vaccine environment by measuring and evaluating the immune response in a population of epidemiological units of production animals, and by detecting and quantifying the shedding effect of animals from air samples. Summary of the Invention

[0012] The present invention relates to a method for determining the environmental immune response to a vaccine in an epidemiological unit of production animals and its assessment, which allows one to understand and report the level of protection of the animals. The method comprises several stages, including: DNA and RNA extraction, analysis of the environmental immune response associated with shedding in animals after vaccination, and notification to the user. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is a diagram of the SIR model of adaptive vaccination (VIP).

[0014] Figure 2 is a chart for monitoring vaccine strains in the environment.

[0015] Figure 3 is a graph showing the immune response of birds by measuring vaccine viruses in their environment.

[0016] Figure 4 It is a tracking chart of the immune system's activity.

[0017] Figure 5 It is the efficiency evaluation diagram of the VIP model (SIR).

[0018] Figure 6 is a graph that tracks peak and fall-off times.

[0019] Figure 7 It is an assessment diagram of interference and protection in the environment. DETAILED DESCRIPTION

[0020] definition

[0021] "Environmental population immunity" in this article refers to measuring and evaluating the immune system's response directly in the environment, without distinguishing between individuals, and measuring the overall population as a unit.

[0022] The "environmental population immune response" (EPIR) in this article refers to the quantitative assessment of vaccine DNA or RNA sequences in air samples caused by animal shedding over time after vaccination.

[0023] "Shedding," as used herein, refers to the secretion of an attenuated pathogen or vaccine after successful replication within the host. This secretion into the environment occurs through secretions or excretions at the mucosal level (respiratory, intestinal, urogenital, etc.).

[0024] The term "epidemiological unit" in this context refers to one or more animal production facilities containing the same batches of animals and having the same hygiene management, husbandry, safety measures, etc.

[0025] "User" in this article refers to individuals or organizations that use the technology described in this article.

[0026] The term "zoonotic epidemiology" in this article refers to the branch of epidemiology that focuses on the study of animal diseases, particularly those occurring in animal populations. It investigates the distribution, risk factors, transmission patterns, and impacts of animal diseases. Zoonotic epidemiology also analyzes the interactions between pathogens, host animals, and the environment, with the goal of better understanding animal diseases and developing appropriate prevention and control strategies.

[0027] "Sample" in this context refers to one or more filters or membranes with controlled pore size obtained after filtration, which are representative of the environment and contain dust particles, microorganisms, trace genetic material, etc. The sample must have corresponding environmental information, such as the sampling time and duration, a description of the sampling environment, the location, the relevant production stage, the batch (or similar batch), the user, and other identifiers that make it unique during the environmental sampling process.

[0028] "Production stage" in this context refers to a stage in the livestock production process, which can be distinguished based on the age of the animals and / or the procedures performed on them.

[0029] "Prediction" in this article refers to the ability to provide health / production status predictions based on data analysis technology.

[0030] The term “departmentalization” in this article refers to projects or departments in the sense of the type of animal production, such as pig fattening, chicken or egg production, or incubators, etc.

[0031] For the purposes of this document, "captive animals" are those animal populations that are housed (usually for commercial purposes) in barns or similar closed or semi-enclosed enclosures during their production phase.

[0032] introduction

[0033] The present invention aims to address the shortcomings of existing vaccine immune response monitoring methods, namely the lack of non-invasive means to provide information on population-specific responses caused by vaccines. Currently, immune response monitoring in animals is performed by drawing blood for representative sampling. This animal blood sampling method is an invasive method and, in some cases, even involves the sacrifice of young animals. In response, the present invention proposes a non-invasive method to determine and assess environmental population immune responses, which is based on the quantification of vaccine DNA or RNA sequences in air samples, reflecting the shedding effect of vaccinated animals.

[0034] This invention redefines the environmental population immune response by quantifying vaccine strain-specific gene sequences in air samples collected from livestock environments over a specific period. This method is uniquely capable of detecting post-vaccination shedding, the excretion of vaccine components from vaccinated animals. Unlike the DIVA (Differentiation of Infected and Vaccinated Animals) approach, which relies on specially designed vaccines and uses serological markers to distinguish infected from vaccinated animals, our innovation is not limited to DIVA vaccines and is applicable to a wider range of immune formulations.

[0035] The present invention is different in that it uses a population-based approach, performed at the barn level, which allows for detailed monitoring of live virus from vaccine strains, providing a collective perspective on the immune response that has not been explored to date.

[0036] The key contribution of this invention is to characterize the dynamics of vaccination responses by interpreting the shape of a curve generated by quantitative data on vaccine viruses in the environment. This curve provides unprecedented information on vaccination efficacy at the population level, allowing for real-time adjustments to immunization strategies to optimize protection against infectious diseases.

[0037] In this context, environmental population immunity refers to directly measuring and assessing the immune system response within the environment, not by individual unit, but rather by making general measurements of the entire population. Therefore, the information obtained from the environment is tailored to the user's needs for facility management, thereby facilitating decision-making.

[0038] The environmental population immune response is a quantitative assessment of temporal changes in vaccine DNA or RNA sequences in air samples caused by shedding from vaccinated animals. Ensuring that the vaccine is administered correctly and at the appropriate time is crucial, and this technology can verify this. Furthermore, this technology allows for specific monitoring to assess vaccine effectiveness.

[0039] The term "shedding" refers to the secretion of pathogens or attenuated vaccines after they have successfully replicated in the host. This secretion occurs through secretions or the excretion of substances from the mucous membranes (respiratory tract, intestinal tract, urogenital system, etc.).

[0040] When pathogens or attenuated vaccines are secreted into the outside world, they can cause infection through direct contact with other susceptible hosts, or indirectly cause infection by being released into the environment (air, water, surfaces of objects), becoming a route of transmission between individuals.

[0041] A typical example is shedding into the air environment through coughing / sneezing or other airway movements, resulting in the dispersion of thousands of particles into the air and the potential for transmission to other susceptible organisms sharing the environment.

[0042] Method Description:

[0043] The approach consists of three stages: determining the environmental population immune response; assessing the environmental population immune response; and generating vaccination response alerts.

[0044] Determination of population immune response

[0045] Given a vaccine A for an epidemic unit, determine its specific concentration (X) in the environment (X A ) is:

[0046] Standardized sampling (at a certain time and protocol) is performed using an air sampling device of the type defined in patent application UY38805. A filter (sample) of dust particles containing microorganisms and associated microbial genetic material is obtained.

[0047] The samples can be stored and transported to the laboratory at room temperature without the addition of stabilizing solution and are stable for up to 72 hours.

[0048] Samples undergo a combination of physical and chemical extraction processes to maximize the amount of genetic material extracted from the filter. These processes may include bead shaking, enzymatic digestion, centrifugation, and / or other validated / assisted extraction methods to ensure efficiency. This phased process improves the sensitivity of subsequent analysis of small samples and ensures representativeness by minimizing bias and maximizing the chance of detecting pathogen diversity in the sample. Typically, when less than 100 μl of DNA or RNA is obtained, it can be allocated for various assays. DNA / RNA quantification is performed using fluorometry and / or absorbance. The information obtained from the sample is expressed in nanograms per cubic meter.

[0049] In one representation, DNA or RNA is extracted from the sample and qPCR is performed using single or multiple fluorescent probes to obtain a Ct value that correlates to the number of copies of A on the farm / facility. Information obtained from the sample X A , in copies / m 3 express.

[0050] Implementing Chart X A (t), to determine the concentration (X A ) as a function of time.

[0051] Environmental population immune response assessment

[0052] Based on the SIR (susceptible, infected, recovered) model adapted to vaccination, we established a derivative model called VIP, such as Figure 1 As shown, the three main populations in the model are the vaccinated population, the vaccinated infected population, and the vaccinated protected population.

[0053] Different indicators were calculated, such as total population (PT), secondary vaccination rate (SVR), vaccine conversion rate (VCR), latent rate (IR), vaccine shedding (VS), vaccine shedding rate (VSR), and measurable vaccine shedding rate (VSRm).

[0054] Total population (TP) : The sum of all populations in the epidemiological unit

[0055] TP=VP+IVP+PP

[0056] in:

[0057] TP is the total population

[0058] VP is the number of inoculated populations

[0059] IVP refers to the number of infected populations that have been vaccinated

[0060] PP refers to the number of protected populations

[0061] Secondary vaccination rate (SVR) : The number of new vaccinations per unit time due to direct or indirect transmission from vaccinated infected populations to vaccinated animals.

[0062]

[0063] in:

[0064] SVR refers to the secondary vaccination rate.

[0065] dIVP = represents the rate of change of the number of vaccinated infectious populations as a function of time.

[0066] dt = time differential

[0067] Vaccine conversion rate (VCR): The number of new individuals fully vaccinated per unit time.

[0068]

[0069] in:

[0070] VCR is the vaccine conversion rate.

[0071] dPP = difference in protected populations over time.

[0072] dt = differential of time

[0073] Incubation rate (IR) : The time required for a vaccinated individual to become infected after first exposure.

[0074]

[0075] in:

[0076] IR is the latency rate

[0077] λ is the vaccine transmission rate

[0078] Vaccine shedding (VS) : Secretion of vaccine into the environment. This refers to the release of vaccine infectious particles from vaccinated populations (or individuals) into the environment, thereby infecting vaccinated people.

[0079] VS∝X A

[0080] in:

[0081] VS stands for vaccine shedding

[0082] X A is measurable vaccine shedding, proportional to VS, X A It is the part that can be analyzed at the environmental level.

[0083] Vaccine Shedding Rate (VSR): Represents the rate of vaccine shedding over time.

[0084]

[0085] in:

[0086] VSR is the vaccine shedding rate

[0087] dVS is the difference in changes in vaccine shedding

[0088] dt is the time differential

[0089] Vaccine shedding rate (VSRm) : Represents the measurable rate of change in vaccine shedding over time.

[0090]

[0091] in:

[0092] VSRm is the measurable vaccine shedding rate

[0093] dX A is the differential that measures the change in vaccine shedding

[0094] dt is the time differential

[0095] Generate vaccination reaction alerts

[0096] Vaccinable animal populations can achieve varying levels of vaccination efficacy following vaccination. While several vaccination conditions are standardized, the outcomes of the vaccination process can be variable, depending on operational, management, or biological conditions. This results in a population's protective response against the pathogen that may be earlier, stronger, more widespread (herd effect), or longer-lasting, depending on the vaccination process used to vaccinate the population.

[0097] We propose that environmental monitoring of vaccine shedding reflects the dynamics of vaccination and can be parameterized to measure the level of vaccination tolerated by closed animal populations.

[0098] In the present invention, it is proposed to generate different alerts based on indicators such as total population (TP), secondary vaccination rate (SVR), vaccine conversion rate (VCR), latent rate (IR), vaccine shedding (VS), vaccine shedding rate (VSR) and measurable vaccine shedding rate (VSRm), including their combinations, such as a) vaccination efficiency, b) vaccine memory efficiency, c) protection obtained by competition with vaccine strains, d) other alerts, which are constructed based on information obtained from the first stage - determination of the environmental population immune response and the second stage - evaluation of the environmental population immune response.

[0099] Here are some of the alerts that may be included in this invention:

[0100] Vaccination efficiency

[0101]

[0102]

[0103]

[0104] Other alerts

[0105] Vaccines are not only a source of protective benefit but can also occasionally cause problems when used alone or in combination, such as revertant vaccine viruses (vaccines carrying new pathogenic mutations) or circulating vaccine pathogenic recombinants (strains that circulate as a hybrid between vaccine and pathogenic strains but are pathogenic, resulting from recombination within certain viral families). Environmental population monitoring of vaccine strain dynamics, combined with sequence identity analysis, can help alert to the risk of these "undercover" strains in vaccine guise.

[0106] Example

[0107] Worked Example 1 : Monitoring of vaccine strains in the environment

[0108] In one embodiment, captive animals are monitored for changes in the load of attenuated vaccine strains in their environment as an indicator of immunity. Live attenuated viruses and bacteria can infect and spread in the environment. In this example, a well-administered vaccination would provide a more intense and shorter peak in the detection of vaccine strains; a mediocre vaccination would flatten the curve because the timing of infection among animals is asynchronous due to immune changes.

[0109] Working Example 2 : Immune responses in birds using environmental vaccine virus curves

[0110] In another example, a series of vaccinations against the same respiratory pathogen in birds with an attenuated virus will produce peaks of vaccine virus in the environment that correspond to the time (weeks) of vaccination. After serial vaccinations, if the animals have an adequate immune response, the peaks of virus in the environment will become lower, flatten, or be delayed because the virus will replicate less in hosts that are better at clearing the virus and, similarly, in populations with more robust immunity. Therefore, by observing the shape of the attenuated virus curve in the environment, we can assess the immune response to the vaccine.

[0111] Predictive Example 1: Tracking Immune System Activity

[0112] Another application of this technology is animal immunity, which involves the activation and repression of genes involved in immune system activity. Traces of this activity can be found in environmental samples. Just as we find pathogens and vaccines of animal origin in the environment, we can also find traces of other animals that may be indicators of animal health.

[0113] In this example, the possibility of finding gene activation / repression groups by qPCR / transcriptomics is explored and patterns will be analyzed in relation to vaccination or infection stage.

[0114] Another application of this example involves searching the DNA for traces of methylation or sequence changes in the environment that correlate with cellular activity involved in the immune response, inflammation, stress, or other physiological indicators. In this way, specific changes associated with the physiological state of the immune system associated with infection or vaccination can be correlated with DNA analysis.

[0115] Predictive Example 2: Graph Efficiency Rating (SIR) using the VIP Model

[0116] Taking the initial vaccination with live attenuated vaccines as an example, for a standard population, using a standardized vaccine dose, a shedding curve should be generated. If vaccination is more synchronized and efficient, the peak of the curve will be higher and occur earlier.

[0117] Graphical descriptors such as peak height, time to peak (from vaccination), shape and path of the shedding curve, and area under the curve are metrics that define vaccination efficiency. Based on these parameters, we can then define vaccination efficiency in percentage terms (the percentage of vaccinated animals that are protected within a given time period) using shedding and VIP model (vaccineable, infective, protected) functions.

[0118] Figure 5 An example of this is the diagram in which animals in different houses vaccinated with more or less efficient vaccines will have different shedding curves, resulting in different changes in the protection status over time (time to reach 50% protection) in each case.

[0119] Predictive Example 3: Tracking Peak and Decline Times

[0120] For monitoring multiple attenuated vaccinations of the same type, in the case of serial vaccinations, the peak of the repeated vaccinations will be more delayed and lower as immunity builds up, making it possible to assign a reference height difference between the "initial peak" and the "repeated vaccinations".

[0121] t(Vx) is the inoculation time, and t(Px) is the shedding peak time corresponding to the inoculation x. Then, when V1, V2, and V3 are successfully inoculated consecutively, the following situation holds: SV(P1)>>SV(P2)>>SV(P3), and t(V1-P1) <t(V2-P2)<t(V3-P3)。

[0122] In a vaccination program administered to animals in a standardized manner, the height and timing of peaks can be standardized and constitute the expected response. If reported deviations from this pattern are significant, this may indicate insufficient dosing, suboptimal spacing, etc., and corrective action can be taken.

[0123] Predictive Example 4: Disturbance and Protection Assessment in the Environment

[0124] Live attenuated vaccines interfere with wild-type strains immunologically and by blocking entry to infection sites through competition for sites (e.g., cell receptors). Certain vaccination programs align with this goal, aiming to provide additional protection during vulnerable periods. For this reason, users may wish to monitor vaccine virus levels in the environment as an indicator of "interference / blocking potential."

[0125] Figure 7 The graph in Figure 2 shows vaccine shedding. In the example of two vaccines, V1 and V2, the area of ​​high protection due to interference is shaded. On the other hand, this overlaps with the protection provided by the vaccines, where each vaccination results in greater protection. Between these two curves, one can visually see areas where the combined protection of vaccination and interference is greater and lesser, allowing one to Planning the use of vaccines within this timeline .

[0126] References

[0127] Hosseini, S., Vázquez-Villegas, P., Rito-Palomares, M., Martinez-Chapa, S.O. (2018). Advantages, disadvantages, and improvements of traditional enzyme-linked immunosorbent assay (ELISA). Springer Briefs in Applied Sciences and Technology (). Springer, Singapore. https: / / doi.org / 10.1007 / 978- 981-10-6766-2_5 .

[0128] Pardo Cobas, MV (November 2006). Epidemiological Compendium.

[0129] National Agricultural University. Reprinted from: https: / / repositorio.una.edu.ni / 2439 / l / nl73p226.pdf .

[0130] "Non-invasive method for diagnosing pulmonary tuberculosis using microdroplets collected from exhaled air." Victor N. Morozov et al. 2018 J. Breath Res. 12 036010. Reprinted from DOI: 10.1088 / 1752-7163 / aab3f2.

[0131] “Mucosal antibodies can be measured in air-dried saliva and fecal samples.” Vetvik H, Grewal HM, Haugen IL, Ahrén C, Haneberg BJ Immunol Methods. 1998 Jun 1;215(1-2):163-72. Reproduced from DOI:10.1016 / s0022-1759(98)00089-1.

[0132] Environmental DNA / RNA for pathogen and parasite detection, monitoring, and ecology. David Bass, Kevin W. Christison, Grant D. Stentiford, Lauren S.J. Cook, and Hanna Hartikainen. Trends in Parasitology, 39, 4, 2023, 285–304. Reprinted from https: / / doi.org / 10.1016 / j.pt.2022.12.010.

Claims

1. A method for measuring and generating alerts on vaccination responses of epidemiological units of production animals, said method comprising determining and evaluating environmental population immune responses from air samples.

2. The method of claim 1, wherein said measuring of said environmental population immune response comprises detecting and quantifying vaccine DNA or RNA fragments resulting from shedding effects in animals following vaccination.

3. The method of claim 2, wherein the alerts generated for vaccination responses of epidemiological units of production animals include alerts for vaccination efficacy, vaccine memory efficacy, protection resulting from competition with vaccine strains, risk of pathogenic reversion to vaccine mutations, and circulating recombinants.

4. The method of claim 2, wherein the environmental population immune response comprises analyzing behavior of numerical values ​​and graphs associated with measurable vaccine shedding "VSm", measurable vaccine shedding change rate "VSRm", and combinations thereof, wherein, "VS" value is expressed in parts / m 3 The "VSRm" value represents the rate of change of the vaccine strain shedding amount "dVS" measured in units of VSR and is obtained by performing qPCR on DNA or RNA extracted from air samples shed by animals after vaccination, and the "VSRm" value represents the rate of change of the vaccine strain shedding amount "dVS" that can be measured within the time "dt" and is expressed as VSRm=dVS / dt.

5. The method of claim 3, wherein the alerts generated for vaccination responses of epidemiological units of production animals include alerts for vaccination efficacy, vaccine memory efficacy, protection resulting from competition with vaccine strains, risk of pathogenic reversion to vaccine mutations, and circulating recombinants.

6. The method of claim 1, wherein the environmental population immune response comprises analyzing behavior of numerical values ​​and graphs associated with measurable vaccine shedding "VSm," measurable vaccine shedding change rate "VSRm," and combinations thereof, wherein, "VS" value is expressed in parts / m 3 The "VSRm" value represents the rate of change of the vaccine strain shedding amount "dVS" measured in units of VSR and is obtained by performing qPCR on DNA or RNA extracted from air samples shed by animals after vaccination, and the "VSRm" value represents the rate of change of the vaccine strain shedding amount "dVS" that can be measured within the time "dt" and is expressed as VSRm=dVS / dt.

7. The method of claim 1, wherein the alerts generated for vaccination responses of epidemiological units of production animals include alerts for vaccination efficacy, vaccine memory efficacy, protection resulting from competition with vaccine strains, risk of pathogenic reversion to vaccine mutations, and circulating recombinants.

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