An OMV Vaccine Derived from a Capsular Group B Meningococcus with Constitutive FetA Expression: Preclinical Evaluation of Immunogenicity and Toxicity

Following the introduction of effective protein-polysaccharide conjugate vaccines against capsular group C meningococcal disease in Europe, meningococci of capsular group B remain a major cause of death and can result in debilitating sequelae. The outer membrane proteins PorA and FetA have previously been shown to induce bactericidal antibodies in humans. Despite considerable antigenic variation among PorA and FetA OMPs in meningococci, systematic molecular epidemiological studies revealed this variation is highly structured so that a limited repertoire of antigenic types is congruent with the hyperinvasive meningococcal lineages that have caused most of the meningococcal disease in Europe in recent decades. Here we describe the development of a prototype vaccine against capsular group B meningococcal infection based on a N. meningitidis isolate genetically engineered to have constitutive expression of the outer membrane protein FetA. Deoxycholate outer membrane vesicles (dOMVs) extracted from cells cultivated in modified Frantz medium contained 21.8% PorA protein, 7.7% FetA protein and 0.03 μg LPS per μg protein (3%). The antibody response to the vaccine was tested in three mouse strains and the toxicological profile of the vaccine was tested in New Zealand white rabbits. Administration of the vaccine, MenPF-1, when given by intramuscular injection on 4 occasions over a 9 week period, was well tolerated in rabbits up to 50 μg/dose, with no evidence of systemic toxicity. These data indicated that the MenPF-1 vaccine had a toxicological profile suitable for testing in a phase I clinical trial.


Final Report
Page 4 Test Facility Study No. 520419 2.

SUMMARY
This study evaluated the potential toxicity and reversibility of reactions to MenPF-1, a prophylactic vaccine for the prevention of infection from bacterial meningitis, when given by intramuscular injection for 4 occasions over a 9 week period to New Zealand White rabbits. In addition, immunogenicity was characterised.
Animals were treated into a hind limb muscle on Days 1, 22, 43 and 64 with necropsy on Days 66 and 92.
The study design was as follows: Text The following parameters and end points were evaluated in this study: viability, clinical signs, injection site reactions, body weights, body weight changes, food consumption, ophthalmology, body temperatures, clinical pathology parameters (haematology, coagulation and clinical chemistry), antibody analysis, gross necropsy findings, organ weights, and histopathological examinations.
There were no unscheduled deaths during the observation period.
There were no systemic signs and no local irritation noted in any animal during the observation period. Body weight and food consumption profiles were unaffected by treatment and there were no eye changes that were considered to be treatment related. There were no differences in body temperatures recorded up to 48 h after injection with MenPF-1.
Other than higher neutrophil numbers, higher fibrinogen levels and minor disturbances in plasma proteins at Day 64 in animals that received MenPF-1, when compared with controls, there were no in-life findings that were considered to be related to treatment with the vaccine.
An increase in titre of specific IgG was observed with increasing dose and over time.
Following completion of 4 week treatment-free period, titres on Day 92 were similar or higher in the majority of animals to those recorded on Day 64.
At Day 66 (2 days after the last injection), 50 µg/dose of MenPF-1 resulted in minor findings at the injection site with foreign material-laden macrophages and giant cells noted. Polymorphonuclear and mononuclear inflammation with myofibre necrosis and/or regeneration, interstitial fibrosis and/or mineralisation were also observed. Lumbar lymph node enlargement was observed at necropsy and this correlated with lymphoid hyperplasia. Accumulation of foreign material-laden macrophages and giant cells was also noted in the lumbar lymph nodes. After a 4 week recovery period, a number of findings persisted in treated injection sites, however were of a lesser severity and frequency.
There were no differences in organ weight that were considered to be related to MenPF-1.
In conclusion, administration of the vaccine, MenPF-1, when given by intramuscular injection for 4 occasions over a 9 week period, was well tolerated in rabbits up to 50 µg/dose.

Final Report
Page 10 Test Facility Study No. 520419 There was only an expected, minor inflammatory response which was associated with vaccine administration, characterised by macrophages, giant cells and polymorphonuclear and mononuclear inflammation at the injection sites with on-going recovery noted. There was no evidence of systemic toxicity.

Final Report
Page 11 Test Facility Study No. 520419

INTRODUCTION
The objective of this study was to determine the potential toxicity of MenPF-1, a prophylactic vaccine for the prevention of infection from bacterial meningitis, when given by intramuscular injection for 4 occasions over a 9 week period to rabbits, and to evaluate the potential reversibility of any findings. Data will support the use of MenPF-1 in humans. In addition, immunogenicity was characterised.
The design of this study was based on the study objectives, the overall product development strategy for the test item, including the following study design guidelines: •  Test and control items were monitored during transit to Charles River, Pre-Clinical Services, Edinburgh. Items were despatched refrigerated (2-8°C); an average temperature of 6.3°C was observed, with a high of 9.3°C which was maintained for 40 minutes. This deviation was considered to be transient and minor and not to have impacted on the integrity of the test and control items.

Test and Control Item Characterisation
The Sponsor provided to the Test Facility documentation of the identity, strength, purity, composition, and stability for the test and control items. A Certificate of Analysis for the vaccine, control item and the bulk vaccine from which the batch for this study was prepared, was provided to the Test Facility and these documents are presented in Appendix 2.
Final Report Page 13 Test Facility Study No. 520419 The vaccine, control item and the bulk vaccine from which the batch for this study was prepared were tested in accordance with Good Manufacturing Practice (GMP).
The Sponsor has appropriate documentation on file concerning the method of synthesis, fabrication or derivation of the test and control items, and this information is available to the appropriate regulatory agencies should it be requested.
No formal expiration dates were provided for the batches of MOX Control or MenPF-1 used on this study. The Sponsor indicated that batches of the control and test items were currently subject to stability testing. Data for the MenPF-1 batch including the latest available timepoint of 3 months were supplied, with further data being generated. The data indicated that at 3 months there was little difference between the results at this timepoint and the results at the initiation of testing. To provide stability data for the period of this preclinical study stability data of 4 months would be required, however, the current data suggested that there would be no reason to expect any degradation in MenPF-1 between 3 and 4 months.
For the MOX control item, current testing is underway, but no data were provided to the Test Facility. Given that this product has been subject to various testing before release, and as this is a control item, the lack of a definitive expiration date was considered not to have had any impact on the data generated from this study.

Reserve Samples
For each batch (lot) of test and control item, a reserve sample of one vial was retained under the appropriate storage conditions by the Test Facility.

Selection, Assignment, and Replacement of Animals
Animals were removed in a random order from their transport boxes and allocated to dose groups on arrival by placing them in separate cages. Cages were housed on racks according to treatment and labelled with the study, animal and group number. Control animals were housed on a separate rack.
A cage plan is presented in Figure 1.
During the week before commencement of dosing, the animals were approved for entry into the experiment on the basis of satisfactory clinical observation records and body weight profiles. There was no replacement of animals.

Disposition
All animals remained on-study until completion of in-life phases at which point designated animals were humanely euthanised by an intravenous overdose of a barbiturate. Details are retained in the study records. 9.7.7. Husbandry 9.7.7.1. Housing Animals were housed individually in stainless steel cages (approximate dimensions 77 x 70 x 48 cm) with a 'Noryl' dual level interior, perforated floor, a mesh top, and a metal food hopper. Beneath each cage was a suspended tray containing absorbent paper. Paper was changed once a week.
Cages, cage racks, hoppers and bottles were changed weekly throughout the course of the study.
Animal room floors and work surfaces were washed daily with disinfectant solution. The ceiling, walls and all other surfaces within the animal room were washed weekly. Cage racks remained in the room throughout washing procedures.

Environmental Conditions
The environmental conditions are continually monitored and recorded every 15 min. Target ranges for temperature and humidity were 16-20°C and 40-85%, respectively, with a room air flow intended to give a minimum of 15 air changes per hour. From animal arrival, until study completion, the average daily ranges for temperature was 15-21°C and for humidity was 33-64% (see protocol deviations in Appendix 1).
Lighting was controlled to provide a 12 h light/dark cycle, normally being 0700-1900 hours.

Food
Harlan Irradiated Certified Global Rabbit Diet, supplied by Harlan, UK, was available ad libitum throughout the study. Each animal was also offered a supplement of hay at least 3 times per week.
Each batch of diet is routinely analysed by the supplier for various nutritional components and chemical and microbiological contaminants.
The results of the diet analysis did not provide evidence of contamination, and so did not prejudice the outcome of the study. Certificates of analysis for each batch used are retained at the Test Facility. The hay is not analysed.

Final Report
Page 16 Test Facility Study No. 520419

Water
Water taken from the public supply (Scottish Water, Edinburgh, Midlothian, UK) was available ad libitum throughout the study.
The quality of water supply is stipulated by Water Quality (Scotland) Regulations 2001 and certificates of analysis for dissolved materials, heavy metals, pesticide residues, pH, nitrates, nitrites and selected bacteria are periodically provided. These analyses are based on water samples taken from these laboratories.
Results of water analysis did not provide evidence of contamination, and so did not prejudice the outcome of the study. Certificates of analysis relevant to the study are retained at the Test Facility.

Animal Enrichment
For environmental enrichment wooden chewsticks, produced by Datesand, Manchester, UK were placed in each cage and treats 'Bunny blocks' as supplied by William Lillico & Son Ltd, UK were also provided.
Analyses of these were considered to indicate that there were no additional substances in sufficient concentration to have any influence on the outcome of the study. Certificates of analysis for these items are retained at Charles River, Edinburgh.

Veterinary Care
All animals were under the care of Charles River's clinical veterinary surgeons, who were available at all times to provide advice and assistance.
On veterinary advice, a lesion to the right hind limb of Animal 27 (Group 3, Recovery Male), which resulted from clipping the injection site, was bathed twice a day for 4 consecutive days from Days 22-25 with aqueous chlorhexidine (an anti-septic). No further advice was required.

Experimental Design
Animals were treated on Days 1, 22, 43 and 64 with necropsy on Days 66 and 92. The Study design was as follows: Text

. Administration of Test and Control Items
The test and control items were administered to the appropriate rabbits by intramuscular injection on Days 1, 22, 43 and 64.
The injection sites (left hind limb -Injection site 1) were clipped free from hair. The aliquots of test and control item were removed from the refrigerator and allowed to warm to room temperature for at least 30 minutes before dosing. To ensure homogeneity, the vials were inverted before dosing and the dose volume required to meet the dosage was administered; Final Report Page 17 Test Facility Study No. 520419 0.5 mL for controls and animals receiving 25 µg/dose or 2 x 0.5 mL µg/dose. The injection sites were delineated.
Animal 27 (Group 3; 50 µg/dose) also received injection in the right hind limb (Injection Site 2) on Day 22. This was due to a small injury caused by clipping of the injection site.
The injection sites were clipped free from hair and delineated before necropsy.

Justification of Route and Dosage Levels
The intramuscular route of administration was selected for this study as this route has been defined by the Sponsor as the route of clinical application/human exposure.
The dose levels were agreed with the Sponsor and took into account the maximum tolerated dose in the test model and other factors such as anticipated therapeutic dose. The test item was produced with similar methodology as for the vaccine product MenBvac (Norwegian Institute of Public Health), based on deoxycholate extracted outer membrane vesicles from Neisseria meningitidis. MenBvac is known to be moderately reactogenic but safe in humans (Nøkleby et al. Vaccine 2007:25:3080-3084).
Clinical injections are planned every 6 to 8 weeks, with three doses intended. In this study, injections were given to rabbits over a shorter period and one more injection (n + 1) was also given. The intended clinical dose may include a dosage of up to 50 µg/dose. This amount was tested in this preclinical study and based on body weight ratio of rabbit 3 kg: human 60 kg and the administration of an additional injection, this was considered to provide adequate safety data.

Definition of Day
The first day of treatment (Day 1) ran from midnight before the first administration until 24 h later, subsequent day numbers (Day 2 etc) also followed this pattern. Body weights and food consumption recorded immediately before dosing on the day of treatment started (Day 1 of the study) were classified as Day 0, relating to the number of days of treatment completed. Subsequent recordings also followed this pattern (that is, body weights, food consumption and clinical signs recorded at the end of the 1 st day of treatment, Day 2 of the study, were documented as Day 1). Any body weights or food consumption recorded before Day 0 were classified as Day -1, etc. Recording of laboratory investigation bleeds and terminal kills were carried out according to study days, that is, Day 1 being the day treatment started.

In-life Procedures, Observations, and Measurements
The in-life procedures, observations, and measurements listed below were performed for all animals.

Mortality/Moribundity Checks
Animals were checked early morning and as late as possible each day for viability.

Detailed Clinical Observations
Once each week, starting during the pretrial period, animals received a detailed clinical examination including appearance, movement and behaviour patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta.

Final Report
Page 18 Test Facility Study No. 520419

Postdose Observations
Animals were examined regularly throughout the day on each dosing day, and once on each non-dosing day. Particular attention was paid to the animals during and for the first hour after dosing. The onset, intensity and duration of any signs were recorded.

Dermal Scoring
Dermal scoring was conducted 0 h (immediately before dosing), 24 h and 48 h after each injection. On 2 separate occasions, scoring was recorded up to 120 h following injections due to erythema observed. Skin was assessed for erythema and eschar formation, oedema formation, skin thickening, desquamation and any other reaction to treatment. The scoring system below was used for assessing erythema, eschar and oedema formation.

Body Weights
Body weights were recorded once during the pretrial period then twice weekly during the dosing and recovery periods.

Food Consumption
The quantity of food consumed by each animal was measured and recorded twice weekly from the beginning of the pretrial period until the end of the study.

Water Consumption
Water consumption was not monitored.

Ophthalmic Examinations
Ophthalmic examinations were carried out on all animals during pretrial and after the completion of dosing. Examinations were conducted by a veterinary surgeon.
The eyes were examined using an indirect ophthalmascope after the application of a mydriatic agent (1% Tropicamide, Mydriacyl  ). The anterior, lenticular and fundic areas were examined. On occasion, repeat samples were collected (where possible) due to clotting of samples. Details are retained in the study records. Values obtained from repeat samples have been reported and used for statistical analysis.

Haematology
Blood samples (0.5 mL) were collected into tubes containing EDTA and analysed for the parameters specified in Text Table 4.
Text A blood smear was prepared from each haematology specimen. Blood smears were labelled, stored and archived. Blood smears were not evaluated as there were no abnormal haematological findings and it was considered that examination would not yield any further information.

Final Report
Page 20 Test Facility Study No. 520419

Coagulation
Blood samples (0.9 mL) were collected into tubes containing 3.8% (w/v) trisodium citrate, processed for plasma, and the plasma analysed for the parameters listed in Text Table 5.   Text Table 5 Coagulation Parameters Activated partial thromboplastin time Fibrinogen Prothrombin time 9.12.1.

Clinical Chemistry
Blood samples (1.5 mL) were collected into tubes containing lithium heparin, processed for serum, and the serum analysed for the parameters specified in Text Table 6. Text

Antibody Sample Collection, Processing and Analysis
Blood samples (2 mL) were collected from an auricular artery once during pretrial, and before dosing on Days 22, 64 and 92. Blood samples were allowed to stand at room temperature for a minimum period of 30 min and processed to serum by centrifugation (at least 1500g at 2-8°C for 10 min).
The serum samples were stored in a freezer set to maintain -80°C and then shipped to the Responsible Scientist on dry ice: Caroline Vipond, Department of Bacteriology, National Institute of Biological Standards and Control (NIBSC), Blance Lane, Potters Bar, South Mimms, Hertfordshire, EN6 3QG, UK.
The samples were to remain frozen throughout transit. Although the temperature was not recorded during transportation, there is evidence that the samples were despatched frozen and were received frozen and in good condition at NIBSC. The immunology laboratory was notified before shipment of the samples, and upon receipt, were stored at ≤-20°C (see protocol deviations in Appendix 1).
The samples were analysed for antibodies against MenPF-1 using a validated ELISA analytical method. No validation was performed for the plate reader software, however, the calibration performed by the service engineer confirmed Operational Qualification (OQ) and standards, and QC samples run with each batch of samples confirmed Performance Qualification (PQ) of the reader.
Any residual anti-therapeutic antibody samples may be retained for research purposes. The results of any subsequent analysis of these samples are not covered in this study.

Unscheduled Deaths
There were no unscheduled deaths during the study.

Scheduled Euthanasia
Main and recovery study animals were euthanised by an intravenous overdose of a barbiturate, weighed and major blood vessels severed to exsanguinate. The animals were euthanised rotating across dose groups such that similar numbers of animals from each group, including controls were necropsied at similar times throughout the day. Animals were not fasted before their scheduled necropsy.

Necropsy
All main and recovery study animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. Scheduled necropsy examinations were conducted by a trained technician and consisted of an external and internal examination and recording of observations for all animals. A veterinary pathologist was available for consultation during normal working hours.

Organ Weights
The organs identified in Text Table 8 were weighed at necropsy for all animals. Paired organs were weighed and are reported together. Organs were weighed before fixation unless otherwise noted. Terminal body weights were used for organ weight analysis.

Tissue Collection and Preservation
Representative samples of the tissues identified in Text Table 9 were collected from all animals and preserved in 10% neutral buffered formalin, unless otherwise indicated.

Histology
Tissues identified in Text Table 9 (except animal identification and bone marrow smears) were embedded in paraffin, sectioned, mounted on glass slides, and stained with haematoxylin and eosin.
Final Report Page 23 Test Facility Study No. 520419 Bone marrow smears were collected at necropsy. The smears were retained but not evaluated.

Histopathology
Histopathological evaluation was performed by a veterinary pathologist with training and experience in laboratory animal pathology.

Peer Review
A pathology peer review was conducted by a second pathologist at Charles River Laboratories, Preclinical Services, Tranent, Edinburgh, EH33 2NE, UK as per the appropriate SOP of the Pathology Department.

COMPUTERISED SYSTEMS
Critical computerised systems used in the study are listed below. All computerised systems used in the conduct of this study have been validated; when a particular system has not satisfied all requirements, appropriate administrative and procedural controls were implemented to assure the quality and integrity of data. The computer systems used by the Responsible Scientist are detailed in the phase report (Appendix 19). Text

STATISTICAL ANALYSIS
All statistical tests were two-sided and performed at the 5% significance level using in-house software. Males and females were analysed separately.
Pairwise comparisons were only performed against the control group (Group 1). The following pairwise comparisons were performed: Control Group vs Group 2 Control Group vs Group 3 Body weight, food consumption, haematology, coagulation and clinical chemistry were analysed for homogeneity of variance using the 'F-Max' test. If the group variances appeared homogenous, a parametric ANOVA was used and pairwise comparisons were made using Fisher's F protected LSD method via Student's t test, i.e. pairwise comparisons were made only if the overall F-test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the nonparametric equivalent of Student's t test).
In circumstances where it was not possible to perform the F-Max test due to zero standard deviation in at least one group, the non-parametric ANOVA results were reported.

Final Report
Page 24 Test Facility Study No. 520419 Organ weights were analysed using ANOVA as above and by analysis of covariance (ANCOVA) using terminal kill body weight as covariate. In addition, organ weights as a percentage of terminal body weight were analysed using ANOVA.
In circumstances where the variances in the ANCOVA remained heterogeneous following log or square root transformations, the data was subjected to rank transformation prior to analysis. Where it was not possible to perform the F-Max test due to the small sample size (less than 3 animals in any group), the untransformed parametric ANCOVA results are reported.
In the ANOVA and ANCOVA summary tables, the results of the analysis are reported indicating the level of statistical significance (p<0.05, p<0.01 and p<0.001) of each pairwise comparison.
Actual p-values are not reported in the summary tables for these analyses.

RETENTION OF RECORDS, SAMPLES, AND SPECIMENS
All study-specific raw data, documentation, samples, specimens and final reports from this study are the property of the Sponsor. These materials will be available at the Test Facility during the progress of the study. When the Final Report is issued, all study-specific raw data, documentation, protocol, samples, specimens and final reports will be archived by the Test Facility for a period of 2 years. After this period, the Sponsor will be contacted to determine the disposition of these materials.
Electronic data generated by the Test Facility will be archived and the software and hardware required to produce it in a readable form will be maintained and available.
All records, and reports generated from phases or segments performed by the Test Site will be returned to the Test Facility for archiving. Residue samples, specimens will be retained at the Test Site for research purposes.

Final Report
Page 25 Test Facility Study No. 520419

Mortality
There were no unscheduled deaths during the observation period.

(Appendices 3 and 4)
There was no signs indicative of systemic toxicity noted during the observation period.
There were local signs recorded at injection sites of 3 animals. Animal 32F (25µg/dose; Group 2) on Days 23-28 had a scab recorded at the injection site and discoloured skin from Days 29-49. Animal 6M (25 µg/dose; Group 2) had a lesion on the injection site on Day 50 and discoloured skin on Day 57 and 64. Animal 27M (50µg/dose; Group 3) had a lesion/scab at the injection site on left hind limb from Days 22-29 and discoloured skin on Day 36.
These local signs were considered minor, transient, had no relationship with dosage and for one animal (Animal 27M) were related to the small injury caused at clipping of injection sites. Overall it was difficult to relate these observations to MenPF-1.

(Appendix 5)
There was no irritation noted at injection sites that were considered to be related to administration of MenPF-1.
The few instances of very slight erythema that were recorded were sporadic, transient and there was no evidence of a relationship with dosage.

Food Consumption
There were occasions where a statistically significant difference in food consumption was recorded. These differences were noted in females receiving 25 µg/dose, where food consumed was lower, when compared with controls (p>0.05). This lower food consumed was recorded on day 45 of the treatment period and Days 73, 87 and 91 of the recovery period. Inspection of the individual animal data indicated that there was individual variation within the data and that 2 animals (Animals 23F and 24F) consumed less food than others within the group. These differences were isolated, did not result in any difference in body weight and there was no evidence of a relationship with dosage. These differences were considered not to be related to administration with MenPF-1.

(Appendix 10)
There were no changes in the eye that were related to administration with MenPF-1.

Final Report
Page 26 Test Facility Study No. 520419

Haematology
(Tables 5-7 and Appendices 13-15) There was an effect on the number of white blood cells in males on Day 66, 48 hours after dosing, that was considered to be related to treatment with MenPF-1.
On Day 66, the number of circulating neutrophils was approximately 2x higher in males receiving 50 µg/dose, when compared with controls (p<0.01). The group mean for the controls was 1.29 x 10 9 /L with an individual range of 0.88-2.34 x 10 9 /L and for the males receiving 50 µg/dose the group mean was 2.43 x 10 9 /L with an individual range of 1.58-3.41 x 10 9 /L. All of the individual values for the males receiving the vaccine were higher than the group mean of the controls and values were also higher than those recorded pretrial. The number of monocytes was higher in males receiving 25 or 50 µg/dose, when compared with controls (p<0.01 or p<0.05, respectively).
On Day 66, the mean cell haemoglobin concentration was higher in females receiving 25µg/dose, when compared with controls (p<0.05). There was no effect on any other red blood cell index and this minor difference was considered not to be related to treatment with MenPF-1.
At Day 92, haematology was considered to be unaffected by treatment with MenPF-1.

Coagulation
( There was also a shorter activated partial thromboplastin time (-8%), which achieved statistical significance, noted in males receiving 50 µg/dose, when compared with controls (p<0.05). One of the control values (Animal 21) was longer than the others in the group and this may be in some part due to this value being from a repeat blood collection. If this value is excluded, inspection of the individual data indicated that although there was variation within the data, broadly between the groups the values were similar. Four of the 6 values recorded for males receiving the vaccine are within the control range. There was no evidence of a relationship with dosage with males receiving 25 µg/dose having a longer activated partial thromboplastin time recorded. This small difference was considered not to be related to treatment with MenPF-1. The shorter prothrombin times recorded in males receiving 25µg/dose and females receiving 50 µg/dose, when compared with controls, was considered to be unrelated to treatment with the vaccine given the small magnitude of change and lack of a relationship with dosage.

Final Report
Page 27 Test Facility Study No. 520419 On Day 92, coagulation was unaffected by treatment with MenPF-1.
On Day 66, globulin (p<0.001) and total protein (p<0.01) was higher in males receiving 25 µg/dose and males and females receiving 50 µg/dose, when compared with controls. There was a lower albumin:globulin ratio in these groups. The protein levels were also higher than those recorded pretrial.
There were other statistically significant differences recorded, for example, lower potassium in males receiving 50 µg/dose, when compared with controls, however these differences were considered to be unrelated to treatment with the vaccine given the small magnitude of change and lack of a relationship with dosage.
On Day 92, there were no plasma chemistry differences that were considered to be related to treatment with MenPF-1. There were statistically significant differences recorded, for example, higher aspartate aminotransferase activity in females receiving 25 µg/dose when compared with controls, however these differences were considered to be unrelated to treatment with the vaccine given the small magnitude of change, similar values recorded pretrial and lack of a relationship with dosage.

(Appendix 19)
The data provided by the Sponsor-designated Responsible Scientist indicated the presence of specific IgG to dosages of 25 or 50 µg MenPF-1/dose. An increase in titre was generally observed with increasing dose and time in both males and females. Although the group mean for each of the groups receiving MenPF-1 was lower on Day 92, inspection of the individual data indicated that 7/12 animals had a similar or higher titre then those recorded on Day 64.
Other gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rabbit, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of MenPF-1.

Scheduled Euthanasia (Day 92)
Test article-related gross findings noted at the terminal euthanasia were not observed at the end of the recovery period. Other gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rabbit, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of MenPF-1.
Final Report Page 28 Test Facility Study No. 520419
On Day 66, absolute adrenal gland weights were statistically higher in females that received 25 or 50 µg/dose, when compared with controls (p<0.05). No dose-response was evident. There was no difference noted after adjustment for terminal body weight and as analysis as a percentage of terminal body weight (relative). This difference was considered not to be related to treatment with MenPF-1.
On Day 92, there were statistically significant differences in males and females that received 25 or 50 µg/dose, when compared with controls; lower absolute and relative liver weight in females (25 µg/dose), lower covariant thymus weight in females (50 µg/dose) and a higher liver weight in females (50 µg/dose). There was non histological correlate and no relationship with dosage, consequently these differences were considered not to be related to treatment with MenPF-1.

Scheduled Euthanasia (Day 66)
There was accumulation of macrophages, observed both in the injection sites and lumbar lymph nodes, which was characterised by aggregates of macrophages containing an abundant, pale basophilic, amorphous cytoplasmic material considered to be aluminium hydroxide. These macrophages were admixed with variable numbers of multinucleated giant cells.
Lymphoid hyperplasia was also recorded which correlated with the enlarged lumbar lymph nodes observed at necropsy.
Other microscopic findings at this dose level observed were considered incidental, of the nature commonly observed in this strain and age of rabbit, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of MenPF-1.
A number of changes were observed in the clinical chemistry, haematology and coagulation group mean values, when compared to their respective controls: there were increased total proteins and globulins, and decreased albumin/globulin ratio in all treated males and in females given 50 µg/dose; increased neutrophil counts in males given 50 µg/dose; increased monocyte counts in all treated male groups; and increased fibrinogen in treated groups from both sexes. These differences correlated with the inflammatory reaction observed in the injection sites.

Scheduled Euthanasia (Day 92)
Some of the microscopic findings noted at the terminal euthanasia (Day 66) were observed at the end of the period off dose (Day 92), however were of a lesser severity and frequency. No treatment related findings were noted in Injection Site 2 (Animal 27).
Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rabbit, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of MenPF-1.

Final Report
Page 29 Test Facility Study No. 520419

DISCUSSION
Intramuscular administration of up to 50 µg/dose of MenPF-1 was associated with an expected, minor physiological response and findings of macrophages, giant cells and polymorphonuclear inflammation at the injection sites and lymph node enlargement at the draining lymph node. These findings were noted 4 weeks after the last injection, however, they were of a lesser severity indicating recovery.
The presence of antibodies to MenPF-1 indicated immunogenicity in rabbits, and confirmed that this species was a suitable selection for this study.
The local inflammatory response noted histologically with the findings of myofibre necrosis and/or regeneration, interstitial fibrosis and/or mineralisation at the injection sites and lymphoid hyperplasia at the injection site draining lymph node correlated systemically with higher fibrinogen and higher levels of the acute phase protein globulin noted after four injections. These minor differences in protein levels are considered to be a physiological response and of little toxicological significance.
The accumulation of the macrophages noted at the injection sites and lymph nodes was considered to be related to the aluminium hydroxide. This response is not unusual where an aluminium based adjuvant has been administered.

Final Report
Page 30 Test Facility Study No. 520419

CONCLUSION
In conclusion, administration of the vaccine, MenPF-1, when given by intramuscular injection for 4 occasions over a 9 week period, was well tolerated in rabbits up to 50 µg/dose. There was only an expected, minor inflammatory response which was associated with vaccine administration, characterised by macrophages, giant cells and polymorphonuclear and mononuclear inflammation at the injection sites with on-going recovery noted. There was no evidence of systemic toxicity.

Final Report
Page 31                                   The target age and weight for the animals at the start of dosing was 12 weeks and 2.5 kg, respectively. At the initiation of dosing animals were approximately 13-14 weeks of age and weighed 2.6-2.8 kg for males and 2.7-3.1 kg for females. The difference between the actual age and weight and the target was considered to be small and rabbits at 13-14 weeks old are still considered to be young adults. This deviation was considered not to have impacted on the outcome or integrity of the study.

Protocol section 11.3 Environmental Acclimatisation
The protocol stated animals would be acclimatised for a period of up to 2 weeks before the first administration. Animals were acclimatised for 15 days before administration of MOX control or MenPF-1 vaccine. This additional day before the start of dosing had no observable effect on the animals and was considered not to have impacted on the outcome or integrity of the study.

Protocol section 12.2 Environmental Conditions
On several occasions the humidity and temperature in the animal room was outside the target range of 16-20°C for temperature and 40-85% humidity. The actual temperature range was 14-22°C, while the humidity range was 29-71%. The environmental deviations did not cause any overt effect in any animal, consequently it was considered that the study outcome was unaffected.