Single Dose Toxicity Study of Polysaccharide VI Conjugate Vaccine in Sprague Dawley Rats Single Dose Toxicity Study of Polysaccharide VI Conjugate Vaccine in Sprague Dawley Rats.

BJSTR. Abstract Typhoid fever continues to be a major public health problem according with estimates of World Health Organization. Conjugation of polysaccharides to an immunogenic protein revert the Thymus independent pattern of polysaccharides to a T-dependent pattern and induce immune response in infants. The aim of this work was to evaluate the toxicological profile a conjugate candidate vaccine against this disease through single dose study in Sprague Dawley rats. Animals were observed daily for local and systemic toxicity symptoms. Also, body weight, water and food consumption, immune response, temperature and local inflammation in the site of injection were evaluated. Gross necropsy was made at the end of the study to each rat, selected organs were weighed, and a full range of tissues was preserved for histological studies. The vaccine not evidenced clinical symptoms, deaths, local effects or adverse systemic toxicological change. Therefore, this vaccine may be considered potentially non-toxic for human. Summary: Typhoid fever remains a major public health problem according to estimates by the World Health Organization. The conjugation of polysaccharides to an immunogenic protein reverses the thymus pattern independent of polysaccharides to a dependent T pattern and induces an immune response in children. The objective of this work was to evaluate the toxicological profile of a conjugate vaccine candidate against this disease, using a single dose trial in Sprague Dawley rats. Animals were observed daily to detect symptoms of local and systemic toxicity. In addition, body weight, food and water consumption, immune response, temperature and local inflammation at the injection site were evaluated. At the conclusion of the study, macroscopic anatomopathology was performed on all animals, selected organs were weighed and all organs processed for histological studies. The vaccine showed no clinical symptoms, deaths, local effects or adverse systemic toxicological changes. Therefore, this vaccine can be considered potentially


Introduction
Salmonella typhi is a Gram-negative bacterium that produce a systemic infection known as typhoid fever. Salmonella enterica serovar Typhi (S. Typhi) continues to be a significant cause of morbidity and mortality in endemic regions. This disease produce annually around of 26 million people are culture positive for S.
The continued burden of typhoid fever and the alarming spread of antibiotic resistant strains lead vaccination as an important control measure. Two typhoid vaccines are widely available, Ty21a (oral) and Vi polysaccharide (parenteral). However, these vaccines are not recommended for use children fewer than 2 years of age because are not efficacy, the group with the highest mortality rate (4% vs 0.4%) compared to the 5-15 years old group [2]. Both typhoid vaccines have other disadvantages; the attenuated strain component of oral Ty21a vaccine requires multiple doses (3 or 4) to induce an effective immune response, which is complex to perform in many developing countries [3]. Moreover, vaccines based on non-conjugated polysaccharides do not induce immunological memory therefore revaccinations is necessary every certain period, and in addition are not effective in infants [4]. It´s well known that conjugation of polysaccharides to carrier proteins produces a reversal in the pattern of response induced thymus independent polysaccharide to a thymus-dependent pattern, which causes the induction of immune response and long-term effective in infants [5]. Newer typhoid conjugate vaccines are at varying stages of development and use. In similar way, Finlay Institute of Vaccine is developing of a new conjugate Vi polysaccharide vaccine for increase his products profile and for contribute in help to prevent typhoid fever. The objective of this study was to perform the preclinical safety testing through the acute toxicity by single dose as part of mandatory aspects before clinical essays.

Animals and Husbandry
Female and male SD rats were purchased from CENPALAB, Rats were provided specialized feed for rodents (ALYco ® ) and the water used was provided in acidified (2.5-2.7 pH) water bottles (750 mL volume), both were available ad libitum. The animal room was maintained at a temperature of 22 ± 2°C and a relative humidity of 55 ± 5%. These parameters were recorded daily in addition to maintaining 12-hour light and dark cycles. Rats were allowed to acclimatize to their surroundings for one week prior to the start of the experimental protocol and were randomly placed into groups of 10. All protocols were approved by the Animal Care Committee and the Biosafety Committee at Finlay Institute of Vaccine.

Vaccine and Experimental Design
Each vaccine vial contained 10 µg of Capsular polysaccharide Vi of Salmonella Typhi per dose (in 0.5 mL, human dose) conjugated to Diphtheria toxoid as active pharmaceutical ingredient and auxiliary substances used as placebo, as show next. The study was designed according to the World Health Organization guidelines on nonclinical evaluation of vaccines and typhoid conjugate vaccines [6,7]. Ten animals were randomly assigned to each group of treatment with the help of a list generated by the Aleator software application (IFV, Habana, Cuba; version 1.2, 1999). The dose was administered intramuscularly, and animals received 4 µg of vaccine dose in a volume of 0.2 mL (40 % of human dose), divided in two sites (both legs). The latter corresponds to the maximum allowable volume according to the chosen administration route and host species [8], which constitutes the reference to establish the upper limit for dose and toxicity in the proposed experimental design. Control animals received phosphate buffer solution (PBS) administered in identical conditions than placebo and vaccinated, who representing the three groups in study. The assay lasted two weeks (14 days).

Consumption
The animals underwent two time at day clinical examinations during first 72 h and then daily with the objective of detecting any behavioral variation or sign of toxicity such as changes in skin and fur, in eyes and mucous membranes or somatomotor activity. At the time of inoculation, rats were weighed to determine their starting weight at the beginning of the study. All rats were identified by the ear punch-out method and weighed at weekly intervals to monitor their weight as a measure of toxicity. Water and food consumption were measured at the start of the experimental design and on alternate days thereafter; daily water and food consumption (mL or g/animal/day respectively) were calculated based on the amount of food and water consumed over the span of a week.

Body Temperature and Muscle Diameter
The body temperature of the animals was measured rectally using a clinical thermometer thin of mercury (Hemeco®, China).
This procedure had a duration of one minute. Muscle volume was measured using a caliper (Scala®, Germany), measuring the diameter of the limb (both legs) before inoculation, both operations were performed at the times 0, 8, 24, 48 and 72 hours post inoculation. These operations were carried out in a similar way to those described above [9].

Hematological and Immunological Studies
A terminal blood sample was taken from Isoflurane -anesthetized rats via cardiac puncture after 6 hours of fasting immediately prior to euthanasia (on day 14). The collected blood was divided in two samples using Eppendorf Tubes® (1.5 mL). One sample was treated with EDTA as Anticoagulant for hematological evaluation.
Another sample was allowed to clot at 4 °C for 30 minutes, and centrifuged at 7000rpm for 10 minutes, and serum was decanted and frozen at -70 °C for immunological test. The following hematological parameters were determined: Red blood cell count, hemoglobin concentration, hematocrit, platelets, leukocytes, lymphocytes, monocytes, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration. A slide smear was made from a single drop of whole blood. Hematological data was generated from an Auto Hematology Analyzer (Model Mindray® BC-2800 Vet., China). Sera collected from rats of 0 and 14 day were analyzed for IgG anti polysaccharide Vi response.
Briefly, ELISA plates were coated 100 µL of 3µg/mL solution

Anatomopathological Studies and Organ Weights
The anatomopathological studies for gross necropsy were were made following WHO guidelines and similar to other studies [6,9,10]. Solid or parenchymal organs (heart, lungs, spleen, liver and kidneys) and thymus were removed, and weights were recorded.
They are expressed as relative weight and were calculated by the following equation: RW = (OW x 100)/EEW, that's means, relative weight (RW) is equal to organ weight (OW) per 100 divided by euthanasia end weight.

Statistical Analysis
Statistical analyses were performed using Graph Pad Prism 5.
Multiwise group analyses were performed using a nonparametric ANOVA with a Dunn's post-hoc test. The significance level was adjusted for multiple comparisons using Bonferroni test. Data were considered significant when p ≤ 0.05.

Results
No mortality or abnormal clinical signs were noted during the study. All of the rats increased their body weight during the 14 days of the study (Figure 1). The weight increase curves of the rats are similar to those observed for this species and in line with the growth curves available from Charles River and the reported in our animals facility [9,10] no statistical differences was observed between vaccine and control groups. We also evaluated the amount of water and food intake (Figures 2 & 3) by the rats over the span of the experimental design, which was not significantly different between any of the groups evaluated in the study for water consumption. (p ≥0.05). However, as usually the males drank more than females on average being 3.45 mL/day vs. 2.43 mL/day showing significantly different between sexes (p ≤0.05). Regarding to food intake, were founded differences between treatment groups for both sexes, the placebo groups, differ from vaccinates and controls (p ≤0.05) during the first 7 days. Nevertheless, the ranges of individual consumption for all groups were between those reported for the species by age and weight [9][10][11]; males consumed more than females on average, being 3.53 g/day for males and females 2.43 g/day. In general, these results were consistent with those observed historical values for rats of this category in our facilities [9][10][11].   Rats don't evidence fever during the study (Figures 4 A), her behavior was in physiological range to the species (CCAC, 2010).
However, there was a significant increase in temperature during the first 8 to 24 hours in the female rats from vaccine group as averaging 37,8 ºC (three female with 38.2 ºC) with respect to control and placebo groups. Although, a similar average was observed for males rats at 8 hours post-vaccine inoculation without difference between groups (p≥0.05). In order to assess the inflammation induced by the vaccine administered intramuscularly, the muscle diameter of the legs was measured before and after receiving the treatments. We saw differences between vaccinated animals with respect to placebos and controls groups for both sexes at 8 hours post-inoculation (p≤0.05) (Figures 4B). In general, the blood parameters assessed was within the physiological ranges described to the species (Table 1) with similarity between sexes and treatment groups. In the series of cells red, although the concentration of hemoglobin in the three groups was slightly higher than that reported in the literature reference [12][13][14] this parameter didn't differ between them and happened similar with white blood cell (Table 2), where we don´t found differences. Immunological response of the rats in our study was similar to previously reported [11]. To both sexes in the vaccine group, the antibody average response (18.01 to female and 13.75 to male) at 14 days was significantly higher (p≤0.05) than the placebo and control groups (0.49 average, Table 3) reinforcing the relevance of the selected animal model and a good immune response to the conjugated polysaccharide Vi vaccine. On the other hand, seroconversion was higher in vaccinated females (90%) than in males (80%). Sex-based differences in immunity have been previously described [11]. The immune factors that regulate the complex immunoendocrine net, and the gender might have a significant function in shaping the immune response and can explain the disparities.  Legend: LEUC-Leucocytes, Lym-Lymphocytes, Mon-Monocytes, Gran-Granulocytes, PLT-Platelets. X, media ± S.D, Standard deviation. Comparison between groups p ≥ 0.05, Kruskall-Wallis The gross necropsy studies performed on all organs and systems for each of the rats studied didn't show any lesions suggesting acute toxicity. Administration sites showed no perceptible local changes. While, some discrete processes of reaction from the immune system at the level of regional lymph nodes near the site of inoculation were observe as histological findings part (Table 4). Such as, the presence of subcapsular secondary follicles in lymph nodes was significant among male rats from vaccinated group respect to controls (p≤0.05). Regarding the relative weight of organs (Table   5), significant differences were observed between heart weight of female's rats and thymus of male's rats, these was between the vaccinated and placebo groups, but not with the control group (p ≤0.05). A joint analysis of the relative weights of the organs, as well as the body weights of the animals under study, led us to conclude that these variables were not affected by the applied treatment.   [9,10,11,13,14]. This parameter have relation with water and food consumptions, as well as health in general.
A relevant indicator for clinical trials is the reactogenicity of the vaccines and the local response [15]. In this sense, temperature and muscle diameter points out that the vaccine has a low reactogenicity, because the local and systemic effects do not exceed

Conflicts of Interest
The authors declare no conflict of interest.