A nanoparticle-based COVID-19 vaccine candidate elicits broad neutralizing antibodies and protects against SARS-CoV-2 infection.

A vaccine candidate to SARS-CoV-2 was constructed by coupling the viral receptor binding domain (RBD) to the surface of the papaya mosaic virus (PapMV) nanoparticle (nano) to generate the RBD-PapMV vaccine. Immunization of mice with the coupled RBD-PapMV vaccine enhanced the antibody titers and the T-cell mediated immune response directed to the RBD antigen as compared to immunization with the non-coupled vaccine formulation (RBD + PapMV nano). Anti-RBD antibodies, generated in vaccinated animals, neutralized SARS-CoV-2 infection in vitro against the ancestral, Delta and the Omicron variants. At last, immunization of mice susceptible to the infection by SARS-CoV-2 (K18-hACE2 transgenic mice) with the RBD-PapMV vaccine induced protection to the ancestral SARS-CoV-2 infectious challenge. The induction of the broad neutralization against SARS-CoV-2 variants induced by the RBD-PapMV vaccine demonstrate the potential of the PapMV vaccine platform in the development of efficient vaccines against viral respiratory infections.

Barriers and facilitators linked to discharge destination following inpatient rehabilitation after traumatic brain injury in older adults: a qualitative study.

PURPOSE: To identify facilitators and barriers associated with returning home for older adults having received inpatient rehabilitation after traumatic brain injury (TBI). METHODS: A qualitative design was used. Five older patients with TBI and four family caregivers were interviewed and six healthcare professionals participated in a focus group. RESULTS: Main facilitators to returning home highlighted by all participants were: (1) Patient's adequate health condition and functional status, (2) Access to health and other services at home, (3) Availability of help from a family caregiver. Conversely, if one of these factors was not met, it represented a barrier. Other facilitators identified were (4) Attachment to one's home, (5) Feeling of commitment toward a loved one, (6) Having the possibility of going through a transitional phase, (7) United front between the patient and the family caregiver towards a return home. Additional barriers to returning home included: (8) Incongruent perspectives, and (9) Unclear knowledge about available health and other services at home. CONCLUSION: The results of this study could be translated into a practical tool to guide patients, families and professionals in the decision about returning home or exploring an alternative option after inpatient rehabilitation for TBI in older adults.IMPLICATIONS FOR REHABILITATIONWhen orienting an older patient home or to an alternative living environment after a traumatic brain injury (TBI), the perspective of rehabilitation professionals can differ from that of patients and caregivers.Professionals tend to emphasize security, whereas patients and caregivers' focus on the well-being associated with home and on the importance of being with their loved one.Integrating the views, values and wishes of older patients with TBI and their caregivers will support a shared decision-making approach for orientation after rehabilitation.

Performance during dual-task walking in a corridor after mild traumatic brain injury: A potential functional marker to assist return-to-function decisions.

Objective: To compare the performance of participants with mTBI and healthy control on locomotor-cognitive dual-tasks in a corridor with limited technology.Design: Prospective study of twenty participants with mTBI (10 women; 22.10 ± 2.97 years; 70.9 ± 22.31 days post-injury), and 20 sex- and age-matched control participants (10 women; 22.55 ± 2.72 years).Methods: Participants performed six different dual-tasks combining locomotor tasks (level-walking, obstacle-crossing, and tandem gait) and cognitive tasks (counting backwards and verbal fluency). Symptoms and neuropsychological performance were also assessed.Results: No differences between groups were found for symptoms and neuropsychological measures. For gait speed, the group effect was not significant, but a significant group X cognitive task interaction was found, revealing a tendency toward slower gait speed in the mTBI group during dual-task conditions. A significantly greater dual-task cost for gait speed was found for the mTBI group. Although no statistically significant differences in cognitive performance were observed during dual-tasks, the mTBI group subjectively reported being significantly less concentrated.Conclusion: The present study revealed that in persons who seem to have well recovered after mTBI, on average 71 days post-injury, alterations in gait are detectable using a simple, "low-tech," corridor-based dual-task walking assessment.

Modulation of Antigen Display on PapMV Nanoparticles Influences Its Immunogenicity.

BACKGROUND: The papaya mosaic virus (PapMV) vaccine platform is a rod-shaped nanoparticle made of the recombinant PapMV coat protein (CP) self-assembled around a noncoding single-stranded RNA (ssRNA) template. The PapMV nanoparticle induces innate immunity through stimulation of the Toll-like receptors (TLR) 7 and 8. The display of the vaccine antigen at the surface of the nanoparticle, associated with the co-stimulation signal via TLR7/8, ensures a strong stimulation of the immune response, which is ideal for the development of candidate vaccines. In this study, we assess the impact of where the peptide antigen is fused, whether at the surface or at the extremities of the nanoparticles, on the immune response directed to that antigen. METHODS: Two different peptides from influenza A virus were used as model antigens. The conserved M2e peptide, derived from the matrix protein 2 was chosen as the B-cell epitope, and a peptide derived from the nucleocapsid was chosen as the cytotoxic T lymphocytes (CTL) epitope. These peptides were coupled at two different positions on the PapMV CP, the N- (PapMV-N) or the C-terminus (PapMV-C), using the transpeptidase activity of Sortase A (SrtA). The immune responses, both humoral and CD8+ T-cell-mediated, directed to the peptide antigens in the two different fusion contexts were analyzed and compared. The impact of coupling density at the surface of the nanoparticle was also investigated. CONCLUSIONS: The results demonstrate that coupling of the peptide antigens at the N-terminus (PapMV-N) of the PapMV CP led to an enhanced immune response to the coupled peptide antigens as compared to coupling to the C-terminus. The difference between the two vaccine platforms is linked to the enhanced capacity of the PapMV-N vaccine platform to stimulate TLR7/8. We also demonstrated that the strength of the immune response increases with the density of coupling at the surface of the nanoparticles.

A Randomized Controlled Study to Evaluate the Safety and Reactogenicity of a Novel rVLP-Based Plant Virus Nanoparticle Adjuvant Combined with Seasonal Trivalent Influenza Vaccine Following Single Immunization in Healthy Adults 18-50 Years of Age.

Inactivated influenza vaccines efficacy is variable and often poor. We conducted a phase 1 trial (NCT02188810), to assess the safety and immunogenicity of a novel nanoparticle Toll-like receptor 7/8 agonist adjuvant (Papaya Mosaic Virus) at different dose levels combined with trivalent influenza vaccine in healthy persons 18-50 years of age. Hemagglutination-inhibition assays, antibody to Influenza A virus nucleoprotein and peripheral blood mononuclear cells for measurement of interferon-gamma ELISPOT response to influenza antigens, Granzyme B and IFNγ:IL-10 ratio were measured. The most common adverse events were transient mild to severe injection site pain and no safety signals were observed. A dose-related adjuvant effect was observed. Geometric mean hemagglutination-inhibition titers increased at day 28 in most groups and waned over time, but fold-antibody responses were poor in all groups. Cell mediated immunity results were consistent with humoral responses. The Papaya Mosaic Virus adjuvant in doses of 30 to 240 µg combined with reduced influenza antigen content was safe with no signals up to 3 years after vaccination. A dose-related adjuvant effect was observed and immunogenicity results suggest that efficacy study should be conducted in influenza antigen-naïve participants.

Increased Immunogenicity of Full-Length Protein Antigens through Sortase-Mediated Coupling on the PapMV Vaccine Platform.

Background: Flexuous rod-shape nanoparticles-made of the coat protein of papaya mosaic virus (PapMV)-provide a promising vaccine platform for the presentation of viral antigens to immune cells. The PapMV nanoparticles can be combined with viral antigens or covalently linked to them. The coupling to PapMV was shown to improve the immune response triggered against peptide antigens (<39 amino acids) but it remains to be tested if large proteins can be coupled to this platform and if the coupling will lead to an immune response improvement. Methods: Two full-length recombinant viral proteins, the influenza nucleoprotein (NP) and the simian immunodeficiency virus group-specific protein antigen (GAG) were coupled to PapMV nanoparticles using sortase A. Mice were immunized with the nanoparticles coupled to the antigens and the immune response directed to the antigens were analyzed by ELISA and ELISPOT. Results: We showed the feasibility of coupling two different full-length proteins (GAG and NP) to the nanoparticle. We also showed that the coupling to PapMV nanoparticles improved significantly the humoral and the cytotoxic T lymphocyte (CTL) immune response to the antigens. Conclusion: This proof of concept demonstrates the versatility and the efficacy of the PapMV vaccine platform in the design of vaccines against viral diseases.

Revealing a decades-old fingermark with cyanoacrylate fuming and rhodamine 6G.

Cyanoacrylate fuming, rhodamine 6G staining and 532 nm laser light visualization were used to reveal a thirty-year-old fingermark on a plastic bag discovered at a double homicide scene.

The quest for a nanoparticle-based vaccine inducing broad protection to influenza viruses.

Influenza virus infections are a significant public threat and the best approach to prevent them is through vaccination. Because of the perpetual changes of circulating influenza strains, the efficacy of influenza vaccines rarely exceeds 50%. To improve the protection efficacy, we have designed a novel vaccine formulation that shows a broad range of protection. The formulation is made of the matrix protein 2 (M2e) and the nucleoprotein (NP) antigens. The multimerization of NP into nanoparticles improved significantly the immune response to NP. The combination of the NP nanoparticles with the PapMV-M2e nanoparticles enhances significantly the immune response directed to NP revealing the adjuvant property of the PapMV platform. The vaccine formulation combining these two types of nanoparticles protects mice from infectious challenges by two different influenza strains (H1N1 and H3N2) and is a promising influenza A vaccine capable to elicit a broad protection.

Activation of innate immunity in primary human cells using a plant virus derived nanoparticle TLR7/8 agonist.

Rod-shaped virus-like nanoparticles (VLNP) made of papaya mosaic virus (PapMV) coat proteins (CP) self-assembled around a single stranded RNA (ssRNA) were showed to be a TLR7 agonist. Their utilization as an immune modulator in cancer immunotherapy was shown to be promising. To establish a clinical relevance in human for PapMV VLNP, we showed that stimulation of human peripheral blood mononuclear cells (PBMC) with VLNP induces the secretion of interferon alpha (IFNα) and other pro-inflammatory cytokines and chemokines. Plasmacytoid dendritic cells (pDCs) were activated and secreted IFN-α upon VLNP exposure. Monocyte-derived dendritic cells upregulate maturation markers and produce IL-6 in response to PapMV VLNP stimulation, which suggests the activation of TLR8. Finally, when co-cultured with NK cells, PapMV induced pDCs promoted the NK cytolytic activity against cancer cells. These data obtained with primary human immune cells further strengthen the clinical relevance of PapMV VLNPs as a cancer immunotherapy agent.

A versatile papaya mosaic virus (PapMV) vaccine platform based on sortase-mediated antigen coupling.

BACKGROUND: Flexuous rod-shaped nanoparticles made of the coat protein (CP) of papaya mosaic virus (PapMV) have been shown to trigger innate immunity through engagement of toll-like receptor 7 (TLR7). PapMV nanoparticles can also serve as a vaccine platform as they can increase the immune response to fused peptide antigens. Although this approach shows great potential, fusion of antigens directly to the CP open reading frame (ORF) is challenging because the fused peptides can alter the structure of the CP and its capacity to self assemble into nanoparticles-a property essential for triggering an efficient immune response to the peptide. This represents a serious limitation to the utility of this approach as fusion of small peptides only is tolerated. RESULTS: We have developed a novel approach in which peptides are fused directly to pre-formed PapMV nanoparticles. This approach is based on the use of a bacterial transpeptidase (sortase A; SrtA) that can attach the peptide directly to the nanoparticle. An engineered PapMV CP harbouring the SrtA recognition motif allows efficient coupling. To refine our engineering, and to predict the efficacy of coupling with SrtA, we modeled the PapMV structure based on the known structure of PapMV CP and on recent reports revealing the structure of two closely related potexviruses: pepino mosaic virus (PepMV) and bamboo mosaic virus (BaMV). We show that SrtA can allow the attachment of long peptides [Influenza M2e peptide (26 amino acids) and the HIV-1 T20 peptide (39 amino acids)] to PapMV nanoparticles. Consistent with our PapMV structural model, we show that around 30% of PapMV CP subunits in each nanoparticle can be fused to the peptide antigen. As predicted, engineered nanoparticles were capable of inducing a strong antibody response to the fused antigen. Finally, in a challenge study with influenza virus, we show that mice vaccinated with PapMV-M2e are protected from infection. CONCLUSIONS: This technology will allow the development of vaccines harbouring long peptides containing several B and/or T cell epitopes that can contribute to a broad and robust protection from infection. The design can be fast, versatile and can be adapted to the development of vaccines for many infectious diseases as well as cancer vaccines.

Using dual task walking as an aid to assess executive dysfunction ecologically in neurological populations: A narrative review.

Within rehabilitation, clinical assessment plays a crucial role in diagnosis, prognostication and making decisions about return to function. The ecological validity of the assessment of executive dysfunction has become a particular focus in neuropsychology and is gaining interest in mobility research and neurological rehabilitation of acquired brain injury or degenerative neurological diseases. In this narrative review, we look at how the task of walking and the inseparable cognitive demands and interference of the surrounding environment are exploited in dual task walking (DTW) paradigms to expose executive dysfunction. While quite a number of studies and reviews have recently focused on the utility of DTW for gait assessment, particularly to assess fall risk, very little consideration has been given to the level of ecological validity required. This paper directly addresses this issue with discussion of evidence and lacunas related to task, personal and technological factors that should be addressed in order to exploit fully DTW paradigms as an ecological assessment tool.

Executive dysfunction following a mild traumatic brain injury revealed in early adolescence with locomotor-cognitive dual-tasks.

OBJECTIVE: To compare gait parameters between children in early adolescence (EA) with and without a mild traumatic brain injury (mTBI) during dual-task walking (DTW). METHODS: Children in EA with mTBI (n = 14; six girls) were compared to those without (n = 13; five girls) while walking in different combinations of obstacle avoidance and cognitive dual-tasks. Gait speed and fluidity and their related dual-task costs (DTC) were analysed along with foot clearance and proximity to the obstacle. RESULTS: No group effects were found for gait speed, proximity or clearance, but were found for fluidity DTC, specifically during the dual Stroop task and when crossing the deeper obstacle. There were also group differences for fluidity during the planning of obstacle avoidance for the narrow obstacle combined with the verbal fluency task and the deep obstacle with no cognitive task. Finally, gait fluidity showed group differences across unobstructed dual-task situations. CONCLUSIONS: Gait fluidity may be a more sensitive variable than gait speed for revealing executive dysfunction following mTBI in EA. Assessing DTW in level walking also seems to show a potential to reveal executive dysfunctions in this age group. These results provide direction for future research on clinical assessment using DTW post-mTBI in adolescents.

Influence of PapMV nanoparticles on the kinetics of the antibody response to flu vaccine.

BACKGROUND: The addition of an adjuvant to a vaccine is a promising approach to increasing strength and immunogenicity towards antigens. Despite the fact that adjuvants have been used in vaccines for decades, their mechanisms of action and their influence on the kinetics of the immune response are still not very well understood. The use of papaya mosaic virus (PapMV) nanoparticles-a novel TLR7 agonist-was recently shown to improve and broaden the immune response directed to trivalent inactivated flu vaccine (TIV) in mice and ferrets. RESULTS: We investigated the capacity of PapMV nanoparticles to increase the speed of the immune response toward TIV. PapMV nanoparticles induced a faster and stronger humoral response to TIV that was measured as early as 5 days post-immunization. The addition of PapMV nanoparticles was shown to speed up the differentiation of B-cells into early plasma cells, and increased the growth of germinal centers in a CD4+ dependent manner. TIV vaccination with PapMV nanoparticles as an adjuvant protected mice against a lethal infection as early as 10 days post-immunization. CONCLUSION: In conclusion, PapMV nanoparticles are able to accelerate a broad humoral response to TIV. This property is of the utmost importance in the field of vaccination, especially in the case of pandemics, where populations need to be protected as soon as possible after vaccination.

Correlates of cognitive functioning in independent elderly patients discharged home from the emergency department after a minor injury.

BACKGROUND: The objective of this study was to explore correlates of cognitive functioning of older adults visiting the emergency department (ED) after a minor injury. METHODS: These results are derived from a large prospective study in three Canadian EDs. Participants were aged ≥ 65 years and independent in basic activities of daily living, visiting the ED for minor injuries and discharged home within 48 hours (those with known dementia, confusion, and delirium were excluded). They completed the Montreal Cognitive Assessment (MoCA). Potential correlates included sociodemographic and injury variables, and measures of psychological and physical health, social support, mobility, falls, and functional status. RESULTS: Multivariate analyses revealed that male sex, age ≥ 85 years, higher depression scores, slower walking speed, and self-reported memory problems were significantly associated with lower baseline MoCA scores. CONCLUSIONS: These characteristics could help ED professionals identify patients who might need additional cognitive evaluations or follow-ups after their passage through the ED. Obtaining information on these characteristics is potentially feasible in the ED context and could help professionals alter favorably elderly's trajectory of care. Since a significant proportion of elderly patients consulting at an ED have cognitive impairment, the ED is an opportunity to prevent functional and cognitive decline.

Engineering of the PapMV vaccine platform with a shortened M2e peptide leads to an effective one dose influenza vaccine.

The emergence of highly virulent influenza strains and the risks of pandemics as well as the limited efficiency of the current seasonal vaccines are important public health concerns. There is a major need for new influenza vaccines that would be broadly cross-protective. The ectodomain of matrix protein 2 (M2e) is highly conserved amongst different influenza strains and could be used as a broad spectrum antigen. To overcome its low immunogenicity we have fused a short peptide epitope derived from the human consensus sequence of M2e (amino acids 6-14, EVETPIRNE) to the N-terminus of papaya mosaic virus coat protein. The fusion harboring coat proteins were assembled around a single stranded RNA into virus-like particles (PapMV-sM2e). The resulting PapMV-sM2e rod-shaped particle was stable and indistinguishable from regular PapMV particles. A single intramuscular immunization with PapMV-sM2e was sufficient to mount appreciable levels of CD4 dependent M2e specific total IgG and IgG2a antibody in mice sera. PapMV-sM2e proved to be self-adjuvanting since the addition of PapMV as an exogenous adjuvant did not result in significantly improved antibody titers. In addition, we confirmed the adjuvant property of PapMV-sM2e using the trivalent inactivated flu vaccine as antigen and demonstrated that the newly engineered nanoparticles areas efficacious as an adjuvant than the original PapMV nanoparticles. Upon infection with a sub-lethal dose of influenza, PapMV-sM2e vaccinated animals were completely protected from virus induced morbidity and mortality. Mice immunized with decreasing amounts of PapMV-sM2e and challenged with a more stringent dose of influenza virus displayed dose-dependent levels of protection. Seventy percent of the mice immunized once with the highest dose of PapMV-sM2e survived the challenged. The survival of the mice correlated mainly with the levels of anti-M2e IgG2a antibodies obtained before the infection. These results demonstrate that PapMV-sM2e can be an important component of a broadly cross-reactive influenza vaccine.

PapMV nanoparticles improve mucosal immune responses to the trivalent inactivated flu vaccine.

BACKGROUND: Trivalent inactivated flu vaccines (TIV) are currently the best means to prevent influenza infections. However, the protection provided by TIV is partial (about 50%) and it is needed to improve the efficacy of protection. Since the respiratory tract is the main site of influenza replications, a vaccine that triggers mucosal immunity in this region can potentially improve protection against this disease. Recently, PapMV nanoparticles used as an adjuvant in a formulation with TIV administered by the subcutaneous route have shown improving the immune response directed to the TIV and protection against an influenza challenge. FINDINGS: In the present study, we showed that intranasal instillation with a formulation containing TIV and PapMV nanoparticles significantly increase the amount of IgG, IgG2a and IgA in lungs of vaccinated mice as compared to mice that received TIV only. Instillation with the adjuvanted formulation leads to a more robust protection against an influenza infection with a strain that is lethal to mice vaccinated with the TIV. CONCLUSIONS: We demonstrate for the first time that PapMV nanoparticles are an effective and potent mucosal adjuvant for vaccination.

Crystal structure of the coat protein of the flexible filamentous papaya mosaic virus.

Papaya mosaic virus (PapMV) is a filamentous plant virus that belongs to the Alphaflexiviridae family. Flexible filamentous viruses have defied more than two decades of effort in fiber diffraction, and no high-resolution structure is available for any member of the Alphaflexiviridae family. Here, we report our structural characterization of PapMV by X-ray crystallography and cryo-electron microscopy three-dimensional reconstruction. We found that PapMV is 135Å in diameter with a helical symmetry of ~10 subunits per turn. Crystal structure of the C-terminal truncated PapMV coat protein (CP) reveals a novel all-helix fold with seven α-helices. Thus, the PapMVCP structure is different from the four-helix-bundle fold of tobacco mosaic virus in which helix bundling dominates the subunit interface in tobacco mosaic virus and conveys rigidity to the rod virus. PapMV CP was crystallized as an asymmetrical dimer in which one protein lassoes the other by the N-terminal peptide. Mutation of residues critical to the inter-subunit lasso interaction abolishes CP polymerization. The crystal structure suggests that PapMV may polymerize via the consecutive N-terminal loop lassoing mechanism. The structure of PapMV will be useful for rational design and engineering of the PapMV nanoparticles into innovative vaccines.

Improvement of the PapMV nanoparticle adjuvant property through an increased of its avidity for the antigen [influenza NP].

The principal caveat of existing influenza vaccine is their failure to provide long-term protection. This lack of efficiency is caused by persistent (drift) and dramatic (shift) antigenic changes on the major surface proteins, the main target of protective immunity generated by traditional vaccines. Alternatively, vaccination with most conserved protein, like the nucleoprotein (NP) can stimulate immunity against multiple serotypes and could potentially provides an extended protection. The NP antigen contains more than 90% protein sequence homology among influenza A isolates and it also contains dominant CTL targets epitopes that made this antigen an attractive target for developing universal vaccine. However, NP protein is a weak antigen and need the use of adjuvant to increase its immunogenicity. We have developed an innovative high avidity VLP (HAV) nanoparticle to improve its adjuvant property to the NP antigen. The nanoparticles are derived from papaya mosaic virus capsid protein (PapMV CP) produced in a bacteria expression system. We generated the HAV by adding an affinity peptide directed to the NP protein at the surface of the VLPs. The fusions of the affinity peptide to PapMV VLPs increased the avidity of PapMV VLPs to NP protein. This modification enhanced the humoral and the IFN-γ response directed to NP. Moreover, the immunity generated by the HAV adjuvanted NP vaccine improved the protection of vaccinated mice to a challenge with influenza virus. The protection was characterized by accelerated virus elimination after the onset of infection and rapid recovery of the vaccinated animals.

Improvement of the trivalent inactivated flu vaccine using PapMV nanoparticles.

Commercial seasonal flu vaccines induce production of antibodies directed mostly towards hemaglutinin (HA). Because HA changes rapidly in the circulating virus, the protection remains partial. Several conserved viral proteins, e.g., nucleocapsid (NP) and matrix proteins (M1), are present in the vaccine, but are not immunogenic. To improve the protection provided by these vaccines, we used nanoparticles made of the coat protein of a plant virus (papaya mosaic virus; PapMV) as an adjuvant. Immunization of mice and ferrets with the adjuvanted formulation increased the magnitude and breadth of the humoral response to NP and to highly conserved regions of HA. They also triggered a cellular mediated immune response to NP and M1, and long-lasting protection in animals challenged with a heterosubtypic influenza strain (WSN/33). Thus, seasonal flu vaccine adjuvanted with PapMV nanoparticles can induce universal protection to influenza, which is a major advancement when facing a pandemic.

[Evaluation of a police strategy for road safety in Quebec: the strategy of the Quebec Association of Police Chiefs]. / Evaluation d'une stratégie policière de sécurité routière au Québec: la stratégie de l'Association des directeurs de police du Québec.

OBJECTIVE: To estimate the effect on collisions of a police traffic safety strategy carried out between January and December 2007, in the province of Quebec, Canada. CONTEXT: This strategy was implemented by several key players (Société de I'Assurance Automobile du Québec, Sûreté du Québec, Montreal Police Department and 34 other municipal police organizations) and targeted the leading causes of traffic casualties such as drinking and driving, speeding and not wearing a seat belt. The strategy has two main components: 1) joint law enforcement operations in which all the police organizations take part, and 2) police organizations targeting local traffic safety problems. Media campaigns supported all operations. INTERVENTION: Over the intervention period, traffic citations issued for speed limit violations, not fastening the seat belt and running the red light or a stop sign increased from 2006 rates by 27, 33 and 8%, respectively. The Société de I'Assurance Automobile du Québec spent more than $5 million in mass media campaigns. RESULTS: According to our results, the strategy was associated with decreases varying between 14 and 36% in collisions with serious injuries. In spite of evidence of downward trends for the period under investigation, neither fatal nor minor injury collisions were significantly affected by introduction of the strategy. CONCLUSION: Police programs characterized by a substantial increase in the distribution of traffic citations and supported by mass media campaigns represent an effective strategy to prevent traffic casualties.