Development of HPV16 mouse and dog models for more accurate prediction of human vaccine efficacy.

BACKGROUND: Animal models are essential to understand the physiopathology of human diseases but also to evaluate new therapies. However, for several diseases there is no appropriate animal model, which complicates the development of effective therapies. HPV infections, responsible for carcinoma cancers, are among these. So far, the lack of relevant animal models has hampered the development of therapeutic vaccines. In this study, we used a candidate therapeutic vaccine named C216, similar to the ProCervix candidate therapeutic vaccine, to validate new mouse and dog HPV preclinical models. ProCervix has shown promising results with classical subcutaneous murine TC-1 cell tumor isografts but has failed in a phase II study. RESULTS: We first generated E7/HPV16 syngeneic transgenic mice in which the expression of the E7 antigen could be switched on through the use of Cre-lox recombination. Non-integrative LentiFlash® viral particles were used to locally deliver Cre mRNA, resulting in E7/HPV16 expression and GFP reporter fluorescence. The expression of E7/HPV16 was monitored by in vivo fluorescence using Cellvizio imaging and by local mRNA expression quantification. In the experimental conditions used, we observed no differences in E7 expression between C216 vaccinated and control groups. To mimic the MHC diversity of humans, E7/HPV16 transgenes were locally delivered by injection of lentiviral particles in the muscle of dogs. Vaccination with C216, tested with two different adjuvants, induced a strong immune response in dogs. However, we detected no relationship between the level of cellular response against E7/HPV16 and the elimination of E7-expressing cells, either by fluorescence or by RT-ddPCR analysis. CONCLUSIONS: In this study, we have developed two animal models, with a genetic design that is easily transposable to different antigens, to validate the efficacy of candidate vaccines. Our results indicate that, despite being immunogenic, the C216 candidate vaccine did not induce a sufficiently strong immune response to eliminate infected cells. Our results are in line with the failure of the ProCervix vaccine that was observed at the end of the phase II clinical trial, reinforcing the relevance of appropriate animal models.

Antipoxvirus Activity Evaluation of Optimized Corroles Based on Development of Autofluorescent ANCHOR Myxoma Virus.

A series of 43 antiviral corrole-based molecules have been tested on myxoma virus (Lausanne-like T1MYXV strain). An autofluorescent MYXV, with an ANCHOR cassette, has been used for the studies. A2B-fluorocorroles display various toxicities, from 40 being very toxic (CC50 = 1.7 µM) to nontoxic 38 (CC50 > 50 µM), whereas A3-fluorocorroles, with one to three fluorine atoms, are not toxic (with the exception of corroles 9, 10, and 22). In vitro, these compounds show a good selectivity index when used alone. Corrole 35 seems to be the most promising compound, which displays a high selectivity index with the lowest IC50. Interestingly, this "Hit" corrole is easy to synthesize in a two-step reaction. Upscaling production up to 25 g has been carried out for in vivo tests. In vivo studies on New Zealand white rabbits infected with myxoma virus show that symptoms are delayed and animal weight is increased upon treatment, while no acute toxicity of the corrole molecule was detected.

Dynamic interactions between cephalexin and macrophages on different Staphylococcus aureus inoculum sizes: a tripartite in vitro model.

BACKGROUND: The bactericidal activity of an antimicrobial drug is generally assessed by in vitro bacterial time-kill experiments which do not include any components of the immune system, even though the innate immunity, the primary host defence, is probably able to kill a large proportion of pathogenic bacteria in immunocompetent patients. We developed an in vitro tripartite model to investigate the joint action of C57Bl/6 murine bone-marrow-derived macrophages and cephalexin on the killing of Staphylococcus aureus. RESULTS: By assessing the bactericidal effects on four bacterial inoculum sizes, we showed that macrophages can cooperate with cephalexin on inoculum sizes lower than 106 CFU/mL and conversely, protect S. aureus from cephalexin killing activity at the highest inoculum size. Cell analysis by flow cytometry revealed that macrophages were rapidly overwhelmed when exposed to large inoculums. Increasing the initial inoculum size from 105 to 107 CFU/mL increased macrophage death and decreased their ability to kill bacteria from six hours after exposure to bacteria. The addition of cephalexin at 16-fold MIC to 105 and 106 CFU/mL inoculums allowed the macrophages to survive and to maintain their bactericidal activity as if they were exposed to a small bacterial inoculum. However, with the highest inoculum size of 107 CFU/mL, the final bacterial counts in the supernatant were higher with macrophages plus cephalexin than with cephalexin alone. CONCLUSIONS: These results suggest that if the bacterial population at the infectious site is low, as potentially encountered in the early stage of infection or at the end of an antimicrobial treatment, the observed cooperation between macrophages and cephalexin could facilitate its control.

Pharmacokinetics of Ertapenem in Sheep (Ovis aries) with Experimentally Induced Urinary Tract Infection.

Sheep are commonly used as animal models for human biomedical research, but descriptions of their use for studying the pharmacokinetics of carbapenem antimicrobials, such as ertapenem, are unavailable. Ertapenem is a critical antimicrobial for human infections, and the description of the pharmacokinetics of this drug is of value for research using ovine as models for human diseases, such as urinary tract infections (UTI). There are currently no ovine models for comparative biomedical research of UTI. The objective of this study was to report the pharmacokinetics of ertapenem in sheep after single and multiple dosing. In addition, we explored the effects of an immunomodulatory drug (Zelnate) on the pharmacokinetics of ertapenem in sheep. Eight healthy ewes (weight, 64.4 ± 7.7 kg) were used in an ovine bacterial cystitis model of human cystitis with Pseudomonas aeruginosa. After disease confirmation, each ewe received 1 g of ertapenem intravenously once every 24 h for 5 administrations. Blood was collected intensively (14 samples) during 24 h after the first and last administration. After multiple-dose administration, the volume of distribution was 84.5 mL/kg, clearance was 116.3 mL/h/kg, T1/2(λz) was 1.1 h, and the extraction ratio was 0.02. No significant differences in pharmacokinetic parameters or time points were found between groups treated with the immunostimulant and controls or after the 1st or 5th administration of ertapenem. No accumulation was noted from previous administration. Our ovine pharmacokinetic findings can be used to evaluate therapeutic strategies for ertapenem use (varying drug dosing schedules and combinations with other antimicrobials or immune modulators) in the context of UTI.

Comparison of hydroalcoholic rubbing and conventional chlorhexidine scrubbing for aseptic skin preparation in dogs.

OBJECTIVE: To compare preparation time, ease of application, and elimination of skin contamination of 3 skin preparation methods for asepsis. STUDY DESIGN: Experimental study. ANIMALS: Healthy dogs (n = 6) with no clinical signs of skin disease. METHODS: Three sites on each dog were randomly allocated to 1 of 3 preparation protocols for asepsis: (1) 5 scrubbings with chlorhexidine gluconate and rinsing (CHXG), (2) washing with mild soap prior to 3 rubbings with hydroalcoholic solution (soap-HAR), or (3) 3 rubbings with hydroalcoholic solution (HAR). The duration of each method of skin preparation was recorded. A Count-Tact agar plate was placed in the center of each site before, immediately after, 1 hour after, and 3 hours after antiseptic application. Plates were cultured, and colony forming units (CFU) were counted. RESULTS: Skin preparation lasted an average of 375 seconds for CHXG, 240 seconds for soap-HAR, and 190 seconds for HAR (P = .00049). Nine CFU (median) were cultured from the skin prior to preparation, with no difference between sites on any animal or for any method. Colony forming units were not detected at any time on any site in any dog after antiseptic application. CONCLUSION: Rubbing with hydroalcoholic (HA) solution was as effective as CHXG and prevented bacterial growth for at least 3 hours under these experimental conditions. Rubbing with hydroalcoholic solution was also faster and easier to perform. CLINICAL SIGNIFICANCE: Because there is currently no known resistance to HA solution, preparation of the surgical site with HAR should be considered to prevent the emergence of bacterial resistance to chlorhexidine as well as potential cross-resistances to antibiotics. Transfer to clinical animals requires additional investigation.

Population Pharmacokinetic Study of Cefazolin Used Prophylactically in Canine Surgery for Susceptibility Testing Breakpoint Determination.

This study aimed to determine the population pharmacokinetic (Pop PK) parameters of cefazolin administered prophylactically at 25 mg/kg intravenously (IV) 30 min before surgery in a canine population of 78 dogs and assess whether covariates, such as sex, age, body weight (BW), breed, health status, creatinine level, and surgery time, have an influence on cefazolin disposition. The ultimate goal was to compute PK/PD cut off values and subsequently establish a specific clinical breakpoint (CBP) for the development of an antimicrobial susceptibility test (AST) of cefazolin in dogs according to the VetCAST approach. Two to 11 blood samples were collected from each dog from 5 to 480 min after cefazolin administration. A two-compartment model was selected, and parameterization was in terms of serum clearance (CL), intercompartmental CL(s) (Q) and volume(s) of distribution (V). The percentage of cefazolin binding to serum protein was 36.2 ± 5.3%. Population primary parameter estimates V1, V2, CL, and Q were (typical value ± SE) 0.116 ± 0.013 L/kg, 0.177 ± 0.011 L/kg, 0.0037 ± 0.0002 L/kg/min, and 0.0103 ± 0.0013 L/kg/min, respectively. Cefazolin presented rapid distribution and elimination half-lives (mean ± SE) 4.17 ± 0.77 min and 57.93 ± 3.11 min, respectively. The overall between-subject variability (BSV) for estimated primary parameters ranged from 36 to 42%, and none of the seven explored covariates were able to reduce this variability by an amplitude clinically relevant. By Monte Carlo simulation, the probability of a PK/PD target attainment (here to achieve a free serum concentration exceeding the MIC for 50% of the dosing interval in 90% of dogs) was computed with a dosage of 25 mg/kg administered IV every 6 h for 4 administrations in 24 h. The computed PK/PD cut off value was 2 mg/L. In conclusion, cefazolin administered prophylactically in surgical dogs at 25 mg/kg IV every 6 h was deemed effective against pathogens with a MIC value ≤ 2 mg/L and from a PK/PD perspective, can be recommended in a wide range of canine patient populations with no necessary dose adjustment for special dog subpopulations.

Laparoscopic-assisted colopexy and sterilization in male dogs: short-term results and physiologic consequences.

OBJECTIVE: To describe laparoscopic-assisted colopexy and sterilization, and evaluate outcome and effects in healthy male dogs. STUDY DESIGN: Experimental study. ANIMALS: Male Beagle dogs (n=7). METHODS: A laparoscopic-assisted, extracorporeally sutured colopexy, and sterilization by ligation and section of the testicular vessels and ductus deferens were evaluated 11 weeks after surgery. Ex vivo tensile tests were performed on the colopexy sites and loss of testicular function was assessed by monitoring serum testosterone, and by ultrasonographic and histologic examinations of the testes. Systemic inflammation and potential iatrogenic colonic functional disorders were investigated by monitoring serum C-reactive protein (CRP) in the perioperative period and from a sulfapyridine (SP) kinetic profile obtained before and 10 weeks after surgery. RESULTS: No intraoperative complications were recorded and clinical outcome was considered fair in all dogs. A mean tensile force of 42 N was required to disrupt colopexies. No relevant postoperative increase in CRP concentrations or changes in SP kinetics were observed. Testicular function was lost. CONCLUSIONS: Laparoscopic-assisted colopexy achieved adhesion of the colon to the abdominal wall and testicular endocrine function and spermatogenesis were eliminated by laparoscopic castration.

Induction of the carrier state in pigeons infected with Salmonella enterica subspecies enterica serovar typhimurium PT99 by treatment with florfenicol: a matter of pharmacokinetics.

Paratyphoid caused by Salmonella enterica subsp. enterica serovar Typhimurium is the main bacterial disease in pigeons. The ability of Salmonella serovar Typhimurium to persist intracellularly inside pigeon macrophages results in the development of chronic carriers, which maintain the infection in the flock. In this study, the effect of drinking-water medication with florfenicol on Salmonella infection in pigeons was examined. The pharmacokinetics of florfenicol in pigeons revealed a relatively high volume of distribution of 2.02 liters/kg of body weight and maximum concentrations in plasma higher than the MICs for the Salmonella strain used (4 microg/ml) but quick clearance of florfenicol due to a short half-life of 1.73 h. Together with highly variable bioavailability and erratic drinking-water uptake, these parameters resulted in the inability to reach a steady-state concentration through the continuous administration of florfenicol in the drinking water. Florfenicol was capable of reducing only moderately the number of intracellular salmonellae in infected pigeon macrophages in vitro. Only at high extracellular concentrations (>16 microg/ml) was a more-than-10-fold reduction of the number of intracellular bacteria noticed. Florfenicol treatment of pigeons via the drinking water from 2 days after experimental inoculation with Salmonella serovar Typhimurium until euthanasia at 16 days postinoculation resulted in a reduction of Salmonella shedding and an improvement in the fecal consistency. However, internal organs in florfenicol-treated pigeons were significantly more heavily colonized than those in untreated pigeons. In conclusion, the oral application of florfenicol for the treatment of pigeon paratyphoid contributes to the development of carrier animals through sub-MIC concentrations in plasma that do not inhibit intracellular persistency.