Cancer esearch rated Systems and Technologies : Mathematical Oncology ilico Modeling and In vivo Efficacy of R cer-Preventive Vaccinations

nloaded cer vaccine feasibility would benefit from reducing the number and duration of vaccinations without ishing efficacy. However, the duration of in vivo studies and the huge number of possible variations cination protocols have discouraged their optimization. In this study, we employed an established model of preventive vaccination using HER-2/neu transgenic mice (BALB-neuT) to validate o–designed protocols that reduce the number of vaccinations and optimize efficacy. With biological g, the in silico model captured the overall in vivo behavior and highlighted certain critical issues. although vaccinations could be reduced in number without sacrificing efficacy, the intensity of early ations was a key determinant of long-term tumor prevention needed for predictive utility in the . Second, after vaccinations ended, older mice exhibited more rapid tumor onset and sharper decline ibody levels than young mice, emphasizing immune aging as a key variable in models of vaccine ols for elderly individuals. Long-term studies confirmed predictions of in silico modeling in which an ne plateau phase, once reached, could be maintained with a reduced number of vaccinations. Furore, that rapid priming in young mice is required for long-term antitumor protection, and that the cy of mathematical modeling of early immune responses is critical. Finally, that the design and ing of cancer vaccines and vaccination protocols must take into account the progressive aging of mune system, by striving to boost immune responses in elderly hosts. Our results show that an the im integrated in vivo–in silico approach could improve both mathematical and biological models of cancer immunoprevention. Cancer Res; 70(20); 7755–63. ©2010 AACR.


Introduction
Tumor immunology compounds the complexity of oncology and of immunology. The interaction creates a huge range of biological variables, what mathematicians would call a "large parameter space." This makes it extremely difficult, if not utterly impossible, to exhaustively test all variables in a given biological experiment, in particular when working in vivo.
Cancer immunoprevention is a recent development of tumor immunology that aims at preventing tumor onset with immunologic means, in particular vaccines (6). Cancer immunoprevention has already proven its potential and worth against human viral tumors (7)(8)(9), the challenge now is to devise strategies to prevent tumors unrelated to viral infections.
Vaccines and other immunologic approaches can indeed prevent tumor onset in cancer-prone, genetically modified animals (6). Proof-of-concept experiments showing that antitumor antibodies and immune-activating cytokines delay the growth of experimental mouse tumors (10,11) were followed by the demonstration that vaccines combining a target antigen with powerful biological adjuvants could arrest aggressive mammary carcinogenesis in mice expressing transgenic oncogenes such as HER-2/neu (6,12). The main challenge issuing from successful experiments in genetically modified mice is now to translate immunoprevention to human situations (6,13,14).
In chemoprevention, the preventive agent must be taken for very long periods, even for the entire life span of the subject, which obviously creates a problem of compliance. Similarly, in the case of vaccines against noninfectious tumors, it was generally found that an effective block of carcinogenesis, yielding long-term tumor-free survival, was only attained when vaccinations were given for the entire life span of the mouse (13).
The long duration of prevention studies, which lasts 1 year or more in mice, further magnifies the complexity of cancer immunoprevention, even in comparison with immunotherapy. It becomes practically impossible to test even a tiny fraction of the relevant biological variables when each experiment lasts 1 year. One clear example is the scheduling of prolonged vaccinations. It is desirable to reduce as much as possible the number of vaccine administrations, in particular, to foreseeably improve compliance and to reduce the risk of side effects in humans; however, the available experimental evidence clearly shows that only chronic vaccination protocols completely protect the host from tumor onset (13). Thus, the problem is to find schedules that operationally minimize the number of vaccinations while maximizing cancer prevention.
To solve this problem, we resorted to mathematical models that faithfully reproduce in silico the behavior of a cancer-preventive vaccination in HER-2/neu transgenic mice (1). We present here for the first time the results of a long-term in vivo cancer immunoprevention study of

Quick Guide to Model and Assumptions
The SimTriplex model The SimTriplex model is an agent-based model specifically tailored to simulate the effects of tumor-preventive cell vaccines in HER-2/neu transgenic mice prone to the development of mammary carcinoma (1). The brief explanation of the model included here is complemented by a more general description in the Supplementary material (Supplementary Text, Supplementary Fig. S1, and Supplementary Tables S1-S4).
SimTriplex includes a variety of cellular and molecular entities, including tumor and vaccine cells, B and plasma cells, helper and cytotoxic T cells, macrophages, dendritic cells, antigens, antibodies, and cytokines (Supplementary Table S1). The attributes of each cell entity include position, age, and state (e.g., resting, activated, memory, antigen-presenting, etc.; Supplementary Table S3). Changes of state (e.g., cell activation, cytotoxicity, cell death, etc.) are governed by a set of rules based on tumor immunology (Supplementary Text and Supplementary Tables S2A and S2B).
Antigen-specific immune interactions [antibody or immunoglobulin/antigen (IG/Ag) and T cell receptor/peptide/MHC (TCR-pMHC)] are modeled with bit-strings (sequences of 0s and 1s). Hamming distances measure the match between bitstrings: 0 matches 1 and vice versa. The probability of an interaction depends on the number of matches.
The simulation space is a two-dimensional triangular lattice (six neighbor sites) with periodic boundary conditions. Cells and molecules are free to move across the lattice sites. At each time step, representing 8 hours of real time, cells and molecules residing on the same lattice site can interact (Supplementary Table S4).
To model the continuous carcinogenic process of HER-2/neu transgenic mice, new tumor cells appear in the lattice, and existing tumor cells replicate (and rarely die). The simulation stops if the total number of tumor cells exceeds a threshold, signifying the formation of a palpable tumor mass, or after a defined number of time steps, typically more than 1 year of real time.
Probabilistic elements affect various starting variables (e.g., initial positions in the lattice) and interactions (e.g., cytotoxic death of tumor cells; Supplementary Tables S2A and S2B). The outcome of each run of the simulator, entailing the generation of a large number of pseudo-casual numbers, is taken to simulate the results of one mouse, thus reproducing experimental variability between individual mice.

Genetic algorithm
A genetic algorithm (2, 3) was used in combination with SimTriplex to search for new vaccination schedules to prevent tumors in HER-2/neu transgenic mice (4,5). Vaccination protocols were encoded as bit-strings of 1,200 bits. Bit positions represented time steps of simulator time, bits set to 1 indicated that a vaccination was to be administered at that time step. The fitness function was based on two fundamental and competing requirements (a) any schedule must yield a mouse survival time of >400 days and (b) the best schedules must have a minimal cardinality. Additional constraints took into account practical issues, e.g., excluding vaccinations during weekends. A set of 80 protocols underwent tournament selection, reproduction used uniform crossover; mutation and elitism were implemented in standard ways. vaccination protocols designed in silico and tested in HER-2/ neu transgenic mice.

Mice
The mammary glands of HER-2/neu transgenic mice on a BALB/c genetic background (BALB-neuT) express a heterozygous mutant rat HER-2/neu transgene under the transcriptional control of a mouse mammary tumor virus promoter (15). BALB-neuT mice display spontaneous mammary carcinogenesis that begins at a very early age with atypical hyperplasia and rapidly progresses to carcinoma in situ, then to invasive carcinoma (15). BALB-neuT mice were bred and maintained in our animal facilities as described (12). Individually tagged virgin females were used for experiments approved by the Institutional Review Board of the University of Bologna.

SimTriplex mathematical model and genetic algorithm
The code of the SimTriplex model (1) was based on the c-ImmSim version 6 implementation of the Celada-Seiden framework (16)(17)(18). To define vaccination protocols, a genetic algorithm used SimTriplex to determine fitness, as previously described (4). See "Quick guide" and Supplementary Material (Supplementary Text, Supplementary Fig. S1 and Supplementary Tables S1-S4).

Cell vaccine
The Triplex vaccine consisted of mammary carcinoma TT12.E2 cells, which express high levels of surface HER-2/ neu gene product p185 and are allogeneic (H-2 q ) with respect to BALB-neuT hosts (H-2 d ), engineered to secrete mouse IL-12 (19). Cells were cultured in DMEM supplemented with 20% fetal bovine serum (Invitrogen) at 37°C in a humidified 5% CO 2 atmosphere. Prior to in vivo administration, cells were treated with 40 μg/mL of mitomycin C (Sigma-Aldrich) to block cell proliferation.

Vaccination and follow-up
The amount of cell vaccine administered per injection was the same for all vaccination protocols and consisted of 2 × 10 6 mitomycin C-treated cells administered i.p. in 0.4 mL PBS. All vaccination protocols started at 6 weeks of age according to different schedules. The Chronic schedule comprised four vaccine administrations over the first 2 weeks of each 4-week cycle (19) up to the 63rd week of age (60 vaccinations); the Early schedule included only the first 12 vaccinations; the Genetic protocol was generated in silico (see Results), aiming to obtain a 50% reduction in the number of vaccinations (hence in the total amount of vaccine used) in comparison with the Chronic protocol; the Heuristic protocol was designed by the authors to spread regularly, over time, the vaccinations of the Genetic protocol. Mice were monitored weekly for mammary tumor onset. Progressively growing masses of >3 mm in mean diameter were regarded as tumors. Mice with tumors in all 10 mammary glands, or one tumor exceeding a mean diameter of 1.5 cm, were killed for ethical reasons. Tumor multiplicity is the number of tumors per mouse at each time point and is expressed as mean ± SEM for each experimental group. Vaccine efficacy on tumor multiplicity was also calculated as the percentage of tumors prevented by each in vivo treatment. Briefly, for each time point, the percentage of efficacy was calculated as (1 − mV/mU) × 100, where mV and mU were the mean multiplicities in untreated and vaccinated groups, respectively; the last value of the untreated curve was used from 36 weeks onwards.

Immunologic monitoring
Mice were bled from a tail vein every 4 weeks starting from the 9th week of age to study the kinetics of peripheral blood cells and antibody levels. Antivaccine antibodies were determined as serum binding to vaccine TT12.E2 cells. Briefly, indirect immunofluorescence was performed with sera (diluted 1:65) and a secondary goat anti-mouse IgG (H + L) chains labeled with AlexaFluor 488 (Molecular Probes). Fluorescence intensity was determined through flow cytometry (FACScan, Becton Dickinson).

Statistical analysis
Kaplan-Meier tumor-free survival curves, built considering the onset of the first mammary tumor, were compared by the Mantel-Haenszel and Gehan-Breslow-Wilcoxon tests, performed with GraphPad Prism 5.01 software. Tumor multiplicities and antibody levels were compared using Student's t test.

In silico design of cancer immunoprevention protocols
The mammary glands of HER-2/neu transgenic BALB-neuT mice are prone to a very aggressive carcinogenic process (Fig. 1A). The Triplex vaccine blocks mammary carcinogenesis when administered to BALB-neuT mice starting at 6 weeks of age, allowing very long (>1 y) tumor-free survival (12). The major limitation of the very effective Triplex vaccine was that only a Chronic protocol (Fig. 1B), entailing more than 60 vaccinations distributed throughout the life of the mouse, blocked mammary carcinogenesis, whereas shorter and/or delayed protocols of only 12 vaccinations eventually left mice exposed to tumor onset ( Fig. 1C; ref. 13).
An exhaustive optimization of the vaccination protocol through trial-and-error (1-year-long in vivo experiments), would have consumed a large number of transgenic mice and a huge amount of time. We then addressed the problem in silico, first with the development of an agent-based model that faithfully reproduced in vivo results ( Fig. 1D; ref. 1), then with the implementation of a mathematical genetic search for reduced, but highly effective, vaccination schedules (4). On the whole, the in silico approach predicted that protocols sparing one half of the vaccine administrations (Genetic protocol in Fig. 1B) could retain most of the efficacy of the Chronic protocol ( Fig. 1C and D). The constraints of the genetic algorithm did not include equal spacing of vaccinations over time, thus resulting in highly aperiodic schedules; to control for this additional variable, we also tested an equivalent periodic vaccination protocol. The protocol designed with the aid of the genetic algorithm is referred to as "Genetic" and the periodic protocol as "Heuristic" (Fig. 1B).

In vivo cancer immunoprevention with vaccination protocols designed in silico
The tumor-free survival curves of vaccinated mice ( Fig.  2A) show that all protocols were successful in prolonging the tumor-free survival of mice (P < 0.0001 versus untreat-ed, Mantel-Haenszel test). The Genetic and Heuristic protocols provided protection from tumor onset similar to that of the Early protocol, whereas the Chronic protocol was more efficient than the Early protocol and kept all mice free from tumors as long as vaccinations were administered (63 weeks of age). Tumor-free survival of the Genetic and Heuristic protocols were not statistically different from that of the Chronic protocol (Mantel-Haenszel), but early tumors were observed before the completion of vaccinations in the former groups (P < 0.05, Gehan-Breslow-Wilcoxon test).

Tumor multiplicity
To better appreciate the effects of various vaccination protocols, we analyzed the number of tumors subsequently appearing in each mouse (tumor multiplicity). It should be noted that a combined analysis of tumor-free survival and tumor multiplicity improves the overall "resolving power" of the system because the latter could uncover significant differences between treatments not readily apparent from tumor-free survival analysis alone (12,19,20).
The results shown in Fig. 2B show clear and significant (see legend) differences in tumor multiplicities at various time points among the Genetic, Heuristic, and Early schedules, which were instead very similar from the point of view of tumor-free survival analysis ( Fig. 2A). In particular, the Genetic vaccination schedule was more potent than both the Heuristic protocol and the Early protocol in reducing the number of tumors appearing over time. On the whole, mice vaccinated with the Genetic schedule had a 60% reduction in the number of mammary tumors at 2 years, similar to that obtained with the Chronic protocol, even though the latter more efficiently controlled the kinetics of tumor development at earlier time points (Fig. 2B). In vivo prevention of BALB-neuT mammary carcinogenesis with in silicodesigned vaccination protocols. A, Kaplan-Meier tumor-free survival curves. The number of mice in each experimental group was untreated, 7; Chronic protocol, 11; Early protocol, 10; Heuristic protocol, 13; Genetic protocol, 12. Statistically significant differences (P < 0.05 at least, Mantel-Haenszel test) were all vaccinated groups versus untreated; Chronic protocol versus Early protocol. B, tumor multiplicity. Points, mean number of tumors per mouse which occurred in the group at the given time; bars, SEM. Significant differences (P < 0.05 at least, Student's t test) were all vaccinated groups versus untreated (at any time); Genetic protocol versus Chronic protocol at 55 to 65 wk; Genetic protocol versus Early protocol from the 65th wk onwards; Genetic protocol versus Heuristic protocol at 35 to 45 wk. C, kinetics of antivaccine antibodies in in vivo experiments. Points, mean of evaluations performed by indirect immunofluorescence of mouse sera on live vaccine cells, followed by flow cytometry; bars, SEM. Gray rectangles depict the thresholds described in the text. Significant differences (P < 0.05 at least, Student's t test) were all vaccinated groups versus untreated (at any time); Genetic protocol versus Chronic protocol at 9 to 17 wk; Genetic protocol versus Early protocol at 9 to 17 wk and from the 29th wk onwards; Genetic protocol versus Heuristic protocol up to 37 wk.

Mechanisms of immune prevention
High-titer antitumor antibodies, in particular against the HER-2/neu surface antigen (15,19), protect Triplex-vaccinated mice from tumor onset, as confirmed also by the lack of protection in antibody-deficient knockout mice (21). Figure 2C shows the kinetics of specific antibodies elicited by the various vaccination protocols. Three features of the antibody response accompanying the Chronic protocol are (a) early vaccinations elicited a rapid and steep increase in antibody titers (compared with the curve of untreated mice); (b) Chronic vaccination maintained a high and steady antibody level; and (c) after the end of vaccinations, antibodies showed a gradual decrease.
The Early schedule, which provided inferior protection from tumor onset, also yielded suboptimal antibody responses as compared with the Chronic protocol: mice receiving the Early schedule had an early and pronounced drop in antibody levels, preceding and paralleling the onset of mammary carcinoma. The response induced by the Heuristic schedule was delayed in comparison with the Chronic protocol, reaching the plateau several weeks later. The Genetic protocol was also less efficient than the Chronic protocol in inducing an early antibody response (compare the first three time points in Fig. 2C, taking into account the logarithmic y-scale).
The considerations made above led us to define two empirical "safety thresholds" (gray bands in Fig. 2C) that could help in predicting the outcome and in immune monitoring: an effective vaccination protocol should first induce a rapid antibody response, which could already be evaluated after the first month of vaccinations, then antibody levels must remain steadily high for the entire life span of the mouse. In the present experiment, whenever either condition was not met, tumor onset invariably ensued. It is interesting to note that the key role of a fast antibody response in cancer immunoprevention was also observed in a similar but distinct transgenic/knockout tumor model (20).
The most likely reason for the suboptimal early induction of antibodies by the Genetic and the Heuristic protocols was that both were less intense in the early cycles than the Chronic protocol. A comparison of Figs. 1B and 2C shows that in the first 3 months of vaccination, the Chronic protocol scheduled 12 vaccinations, the Genetic protocol 8, and the Heuristic protocol 6, proportional to the ensuing antibody levels (Chronic protocol > Genetic protocol > Heuristic protocol), and eventually to protection from tumors (Fig. 2B).

Immune aging
The decrease in antibody levels after 1 year of age in the Chronic, Genetic, and Heuristic protocols could be caused either by immune exhaustion elicited by intensive immune stimulation or by the natural aging of the immune system. We did not detect temporal variations in the expression of exhaustion-related receptors PD-1 and PD-L1 (22) in mice receiving the very intense Chronic protocol (data not shown).
A relevant result of the in vivo experiment came from the follow-up of the Chronic protocol after the last vaccination: mice remained tumor-free for several weeks, but eventually, all mice succumbed to tumors ( Fig. 2A). A comparison of the time span between the last vaccination and the median duration of tumor-free survival in the Early and Chronic protocols (Fig. 3) shows that the interval was much shorter in the Chronic protocol (15 weeks) than in the Early protocol (37 weeks). A possible cause is the natural aging of the immune system (23). Figure 4 shows a direct comparison of the survival curves predicted in silico or observed in vivo. Figure 4A shows schedules that were repeated in the present experiment as controls, replicating the results originally used for the tuning of the simulator, whereas Fig. 4B shows the tumor-free survival curves obtained with vaccination schedules tested for the first time in vivo. It seems that the main divergence was that the simulator predicted a less rapid decline of tumor-free survival curves for the Genetic and Heuristic protocols, the same applies to the Chronic protocol for time points beyond the end of vaccinations. With regard to the untreated and Early protocols (Fig. 4A), in vivo experiments yielded a faithful replicate of previous ones, providing an excellent alignment between predicted and actual curves.

In vivo results versus in silico prediction
The relative potency of the various protocols revealed by this analysis of in vivo results is therefore similar to that predicted by the simulator in silico. To provide a visual comparison, we devised a transform of tumor multiplicity allowing side-by-side plotting with predicted survival curves. The transform estimates the percentage of tumors prevented at a given time point by each treatment, taking mean tumor multiplicity of untreated mice as 100%. Figure 4C shows that the percentage of prevented tumors fit the predictions of the simulator better than tumor-free survival curves (compare with Fig. 4B).
The scale of the variable measuring antibody levels in the simulator was roughly similar to the scale of cytofluorometric measures, therefore allowing visual comparisons of overall trends. The long-term decrease in antibody levels observed in vivo (Fig. 2C) was mirrored by that predicted by the  simulator (Fig. 4D). It is interesting to note a good agreement in the long-term behavior of the curves, which was evident, for example, in the case of the Early protocol.

Discussion
Mathematical models of cancer in many instances are fed by data from in vivo experiments. Too frequently, the development of clever biomathematical models, based on sound biological data, is not followed by an extensive biological validation, and the model fails to find widespread application where it is most needed, i.e., in the biomedical field (24). We showed here that a faithful agent-based mathematical model, coupled with a well-constrained genetic algorithm yields effective cancer-preventive vaccination protocols competitive with human-designed protocols. Because the model cannot include everything, it is likely that what is missing from the model determines the different degrees of qualitative versus quantitative prediction. It is worth noting that human knowledge in this area of tumor immunology is still poorly systematized, therefore, the interaction of mathematical and biological model systems yields various insights for future developments both in silico and in vivo (25)(26)(27).
Testing in long-term (2 y) in vivo experiments allowed us to precisely assess the predictions obtained in silico and to pinpoint specific aspects of the mathematical model and algorithms in need of further refinement. The major divergence from the predictions was that the efficacy of Genetic and Heuristic schedules was overestimated by the simulator. In vivo results of all other schedules faithfully reproduced those obtained in previous experiments as well as values previously used for simulator tuning, eventually mirroring the predictions. Long-term in vivo results of the Genetic and Heuristic vaccination protocols could then be used for further tuning of the mathematical model and to verify if variables show significant changes, i.e., if the computer model is stable.
The study of in vivo results offers hints that go beyond the mere tuning of simulator variables. In fact, analysis of tumor multiplicity showed that the simulator-designed Genetic schedule indeed prevented more tumors than the Heuristic and Early schedules, and the kinetics of prevented tumors was in better agreement with the predicted efficacy of the Genetic schedule than that of tumor-free survival. This agreement implies that simulator results embed a richer information than tumor-free survival curves; however, it is clear that only a multiorgan implementation of the simulator could better mirror the biological system and improve predictive ability.
A traditional practice of vaccinology is to schedule periodic vaccinations with regular and symmetrical intervals. In our mice, the highly aperiodic Genetic protocol was as effective as (tumor-free survival), or even more effective than (tumor multiplicity) the equivalent periodic Heuristic protocol. Therefore, periodicity per se was not required for an efficient stimulation of the immune system, whereas the temporal distribution of vaccinations played a decisive role. As predicted in silico, many vaccinations of the Chronic protocol were redundant in the immune plateau phase and can be avoided in the future.
We did not place constraints on the genetic algorithm with regard to the distribution of vaccinations over time periods (i.e., initial priming vaccinations versus later boosts), but analysis of in vivo results clearly showed that vaccination density in the priming phase, and the ensuing intensity of the elicited immune response, was a major determinant of long-term protection of mice from tumor onset, thus corroborating in HER-2/neu transgenic mice analogous conclusions obtained in a different, bigenic model of cancer immunoprevention (20).
These results provide at once useful indications for the implementation of ad hoc constraints in silico, and for immediate in vivo studies of novel, intensified vaccination protocols with more vaccine administrations in the initial phases than are currently scheduled in the Chronic and Early vaccination protocols.
Early antibody responses observed in vivo correlated with protection from tumor onset and predicted tumor-free survival, strongly supporting the need for intense protocols during the early cycles of vaccination. The strong correlation between early antibody response and long-term tumor-free survival could be exploited in the future to perform shorter in vivo experiments to test new vaccination schedules in just 3 to 4 months. Long-term in vivo experiments of the type shown here could then be performed only to obtain comprehensive evaluations of promising new protocols after successful short-term testing.
The kinetics of antibody response was not originally used to tune the mathematical model, therefore, the similarity between in vivo (Fig. 2C) and in silico (Fig. 4C) results was remarkable. However, some of the differences between vaccination protocols observed in vivo were less apparent in antibody numbers in silico. Therefore, a further suggestion for improvement of the model is to use immune response variables, in particular antibody titers, to seek a better correlation with the corresponding in vivo results and with predicted tumor-free survival.
The cancer immunoprevention protocols studied here allowed a better appraisal of the role of immune aging, a very important aspect of vaccine response that is too frequently either ignored or simply taken for granted. Moreover, most preclinical studies are performed in young hosts vaccinated for short periods of time, whereas both cancer immunoprevention and cancer immunotherapy need to be applied to adult and aging humans and for very long time spans.
After reaching an absolute maximum between 20 and 30 weeks of age, antibody responses steadily declined even in mice undergoing boosting vaccinations (see Chronic, Genetic, and Heuristic protocols in Fig. 2C), thus indicating that immune aging lowered the maximal immune responses that could be attained with a powerful vaccine.
In previous studies, long-term vaccination experiments were either terminated at 1 year, or in a few instances, vaccination cycles continued for the entire life span of the mouse, but in the present work, we followed for the first time a group of aging mice after the end of immunizations at 1 year of age. The most striking result was the shortening of the tumor-free period, which was cut by more than half in comparison with young mice (Fig. 3), suggesting a dramatic decline in duration and efficacy of vaccine-induced immune memory in aged mice. Recent intervention studies indicate that immune aging could be delayed (28) or overcome with specific modifications of vaccine components (29), and we are currently investigating whether modified vaccines can indeed prolong cancer prevention in aged hosts.
For the development of refined mathematical models, both in cancer immunity and in biology at large, our results clearly indicate that all long-term models should explicitly take into account the natural aging of the host to avoid optimistic overestimates when applied to temporal frameworks that could entail age-related declines in physiologic and homeostatic phenomena. These findings could lead to synergistic improvements in both mathematical and biological models of cancer immunoprevention.