An innovative method of nonclinical efficacy and toxicological evaluation of recombinant Salmonella typhi Ty21a expressing HPV16 and 18 L1 proteins

Advancement in technology led to development of live attenuated Salmonella typhi Ty21a as enteric vector for expression of foreign proteins. Such vector platform is inevitable for development of vaccine candidate against human papilloma virus (HPV), the etiological agent of cervical cancer with high prevalence in developing nations. The high risk HPVs like type 16 and 18 contributes to 70% of cervical cancer, hence Indian Immunologicals Limited (IIL), Hyderabad, India developed a recombinant HPV vaccine by introducing HPV 16 and 18 L1 protein coding genes into attenuated S. typhi Ty21a vector. Being a genetically engineered enteric vector vaccine, it would be less expensive, with an ease of oral administration, instead of injectable that needs trained personale, is an added advantage for low socioeconomic setup compared to existing HPV vaccines. Establishing the nonclinical efficacy and safety/toxicity as per the national/international regulatory guidelines has become major constrain for such recombinant S. typhi HPV (rSt.HPV) vaccine. Since, the intended clinical mode of rSt.HPV is through oral route, whereas the live attenuated S. typhi Ty21a doesn't colonize in gut of laboratory animals to be used for nonclinical experiments. Hence, an alternate and unconventional method of ‘intranasal drug testing’, was followed for nonclinical efficacy and toxicity evaluations. An array of parameters specified by regulatory agencies were investigated in mice, rat and rabbits administered with rSt.HPV through, intra-peritoneal, intranasal and oral routes, the intended clinical route.• Current unconventional and innovative nonclinical testing procedures helps in exploring the alternate methods by pharmacologist/toxicologist.• Ultimately, such new drugs developed through technology must serve the humankind justifying the guidelines of regulatory agencies.


Method Details
Back ground of method development Current modified method was originally reported by Nardelli-Haefliger et al. [1] to demonstrate the immune response evaluation against HPV 16 L1 proteins expressed through live attenuated Salmonella typhimurium CS022 vector. The S. typhimurium was administered to mice through oral and nasal routes. However, an advancement in technology over a period led to fine tune such recombinant vaccines in various aspects viz. selection of live attenuated vectors which colonize in human gut, in vivo construct stability, constitutive expression system, markers other than antibiotic resistance etc.. In view of the mentioned properties and the expertise in development of recombinant vaccines, the M/s. Indian Immunologicals Limited (IIL), Hyderabad, India has developed a proprietary anti-HPV vaccine candidate by introducing the codon optimized HPV 16 and 18 L1 protein coding genes into live attenuated Salmonella typhi Ty21a as vector, as the HPV 16 and 18 together responsible for 70% of cervical cancers caused throughout the world [2][3][4] .
The commercially available vaccines against HPV are expensive, being the injectable needed the cold-chain system and trained personale for administration. In addition these vaccines were known to have the adverse effects such as headache, gastrointestinal disturbances, syncope, myalgia, fever, etc. Whereas, the recombinant S. typhi Ty21a expressing HPV 16 and 18 L1 proteins (r.St.HPV) developed by IIL, Hyderabad, India has advantages over existing vaccines in terms of its cost-effectiveness, admissible through oral route hence doesn't required sophisticated infrastructure thus better suits for low-socioeconomic countries. It is noteworthy to mention here that a higher prevalence of HPV related cervical cancers were reported from developing and low-socio economic countries with a mortality rate of 88% [5] .
The current innovative method confines for establishing the nonclinical efficacy and safety/toxicity as per the national/international regulatory guidelines. The major constrain is the inability of live attenuated S. typhi Ty21a colonization in gut of laboratory animals when administered through oral route, which is the intended clinical route of vaccination, hence nonclinical efficacy and safety/toxicity studies of r.St.HPV has become challenging [6] . Hence, an alternate and unconventional method of 'intranasal drug testing', was followed for nonclinical efficacy and toxicity evaluations. An array of parameters specified by regulatory agencies were investigated in mice, rat and rabbits, which were administered with rSt.HPV through intra-peritoneal, intranasal and oral routes, the intended clinical route [7] .

Chemicals, Reagents & sterile-ware
1. Standard sterile pellet diets comprised of all macro and micronutrients ( Tables 1 and 2 ) according to mice, rat and rabbits requirements were obtained from NCLAS, ICMR-NIN, Hyderabad, India [8] . 2. In addition to standard pellet diet, rabbits were given 'Lucerne grasses' and Sprouted Bengal gram [8] . 3. Diet and purified water collected through activated charcoal filter and subsequently exposed to UV light were given as ad libitum to animals.

Procedure
Preparation & Administration of Anastasia 1. The ingredients of 'anesthesia', their role and composition were given in Table 3 . 2. Ketamine hydrochloride was collected into sterile graduated tube and then added the Xylaxine hydrochloride, followed by gentle mixing of the contents. 3. The Diazepam was added to Ketamine-Xylaxine mixture. 4. Anesthesia solution was mixed gently and brought the final concentration through addition of sterile distilled water. 5. The tubes were labelled and placed in Ice pack gels, until administration. 6. Body weight of animals to be anesthetized were recorded and the anesthesia collected accordingly into '1.0 mL' sterile syringes. 7. Animal (mice & rat) brought to 'supine position' by holding tail of 'prone position' animal with one hand and dorsal-neck skin with other hand, followed by tilting the animal 180 o across the plane. 8. The lower quadrant of abdominal area (fur shaved region) was whipped with 70% IPA swabs, and subsequently allowed to dry. 9. A volume of (75-100 μL / mice and 750-10 0 0 μL / rat) of anesthesia solution was administered to animals according to their body weights through intra-peritoneal route. 10. Delivery of anesthesia was ensured by introducing the syringe into lower abdominal skin with an angle of ~10 °, to avoid accidental penetration into viscera. 11. Rabbits were administered 1.5-2.0 mL of anesthesia through intramuscular route (fur shaved region of hind limb thigh, whiped with 70% IPA) according to body weight. 12. The unconscious stage of anesthetized animals were achieved at 5-10 min of post-exposure of anesthesia and persist for 20-30 min. 13. Anesthetized animals were laid-down in supine position with their heads raised using bedding husk ( Fig. 1 A,

Test Compound Suspension Preparation
1. Lyophilized powder of attenuated S. typhi Ty21a and recombinant S. typhi Ty21a expressing HPV 16 and 18 L1 proteins (rSt.HPV) were obtained from Indian Immunologicals Limited (IIL), Hyderabad, India. 2. The lyophilized powders of S. typhi and rSt.HPV were diluted in sterile PBS under aseptic conditions. 3. Bacterial concentrations of suspension were determined by plating three successive dilutions in duplicate on Salmonella-Shigella agar. 4. The bacterial colonies were enumerated after incubation at 37 °C and expressed as 'colony forming units' (CFU) /mL. 5. Stock bacterial suspensions were prepared according to previous step and diluted to highest doses of 50xPD, 10xPD, AD (5xPD) and PD of mice, rat and rabbits.
Bacterial suspension Administration 1. The acute testing of live attenuated S. typhi Ty21a expressing HPV 16 and 18 L1 proteins was performed in mice and rats by single administration of 10xPD and 50xPD through intranasal route. 2. Sterile filtered tip fitted micro-pipets were loaded with the designated volumes of bacterial suspension according to the body weights of animals. 3. The volumes not greater than 20 μL and 100 μL were administered to anesthetized unconscious stage, supine positioned mice and rats, respectively through right nostril ( Fig. 1B and 2B ). Whereas, for rabbits a volume of not greater than 200 μL of vaccine was administered through right nostril under anesthetized condition (Fig. 3B) . 4. After test compound administration, animals on husk bedding were monitored until they get consciousness, without disturbing. 5. Similarly, the sub-chronic toxicity testing of rSt.HPV vaccine was undertaken in rats and rabbits with three successive dosing on 1 st , 3 rd and 5 th day by administration of two concentrations of vaccine i.e., PD and AD (5xPD). 6. In sub-chronic toxicity testing, the prophylactic doses (PD) of vaccine was administered through intranasal routes to anesthetized rats and rabbits. In addition to intranasal route, the prophylactic dose effect of vaccine was assessed through oral route in rats and rabbits. 7. The average dose (AD = 5xPD) effect of vaccine was undertaken through intranasal route only in rats and rabbits, as mentioned above. 8. The allergenic and immunogenicity potential of the r.St.HPV vaccine was assessed by administration of test compound through intra-peritoneal (IP) and intra-nasal (IN) routes in mice. Mice were primed with 250 μL of vaccine (PD and AD (5xPD)) by administration through intra-peritoneal route after whipping the local area with 70% IPA. Similarly, the booster dose was administered on 7 th day. 9. Intranasal route of allergenic and immunogenicity evaluation was undertaken in mice, anesthetized as explained above. Anesthetized mice were primed with 20 μL of vaccine (PD and AD (5xPD)) administration through IN route on 0 th day following the 'step no. 2-4'. Subsequently the mice were anesthetized and boosted with vaccine on 28 th and 56 th days through IN route. 10. All the animals were observed and investigated for below mentioned parameters until the end of the experimental period as per the regulatory guidelines.

Nonclinical Efficacy and Safety Studies Undertaken
Acute Toxicity Testing 1. The mice and rat administered with single exposure of 10xPD and 50xPD doses of vaccine were observed for morbidity and pre-terminal mortality on 1 st , 2 nd , 3 rd , 6 th , 12 th and 24 th hour of post-vaccine exposure. 2. Later on the animals were observed for 14 days for cage side activities along with recording of bodyweights bi-weekly. 3. The food and water intake was recorded daily. 4. In case of pre-terminal mortality, autopsy was carried out to unveil the test compound effect if any on vital organs, otherwise all the animals were euthanized on 14 th day by examining the animals for gross necropsy and collecting the vital organs for archiving.  Allergenic and Immunogenicity Testing of rSt.HPV 1. The nonclinical efficacy evaluation of test compound i.e. live attenuated recombinant S. typhi Ty21a expressing HPV 16 and 18 L1 protens (rSt.HPV), was undertaken in mice through two different routes. 2. Allergenic profile of vaccine, if any was assessed by administration of Ovalbumin (2% OVA) to mice (positive control) and quantification of specific IgE levels in serum. 3. The PD and AD were administered through intra-peritoneal routes on 0 th day and one booster dose was administered on 7 th day. Serum anti-HPV 16 and 18 specific IgG and IgE levels were determined on 0 th , 14 th and 28 th day. Half of the animals were euthanized on day 14 th and remaining on 28 th day to unveil the test compound exposure effect on various organs systems. 4. Bacterial suspension of prophylactic dose (PD) and average dose (AD) were administered to anesthetized mice through intra-nasal route as explained under above sections. The animals were primed with test compound on 0 th day and subsequently boosted on 28 th and 56 th days. Blood was collected on 0 th , 28 th , 56 th , 63 rd and 78 th days to assess anti-HPV 16 and 18 specific IgG and IgE levels. At the end of experiment mice were euthanized and collected the vital organs/tissues for histopathological examination.

Observations and Parameters Investigated and Analyzed
The animals were observed for morbidity and mortality as per the regulatory guidelines. The following observations, bio-sample collection and analysis of various parameters were undertaken to unveil the 'vaccine administration effect', if any.

Functional observation battery (Safety pharmacology)
Live phase of animals. The animals were observed daily for 'behavioral activities' viz., active, nonactive, partially active and hyperactive. The water and food intake was monitored daily along with recording of body weights on alternate days.
Cage side observations. The vaccine exposed animals were monitored for their home cage activity viz., lying on side, resting, and alertness were monitored daily. The excretion of urine and fecal matter amount, colour and consistency were noted daily. In addition, the behavior of the animals while removing from cage was recorded whether the animals were quiet easily removed / runs around in the cage / oriented towards the investigator/aggressive or any vocalization.
Physical Examination. Periodically the animals were observed for physical appearance of hair coat, piloerection, lacrimation, salivation, respiration character and rate, along with eye prominence and eye lid closure. In addition, the biting behavior, convulsions and tremors if any, were examined daily.
Neurological Examination. The effect of vaccine administration on neurological functions were assessed in terms of locomotor activity, rearing activity, tail elevation, static limb position, abnormal & ataxic gait, head position and pinna touch response twice in a week.
Allergenicity. Animals administered with vaccine were examined daily for the allergenic effect of test compound, if any, in terms of skin reactions and hair loss, along with watering and congestion of eyes.
Sleeping time during anesthesia. In order to intranasal administration of vaccine, the animals were anesthetized. Therefore sleeping time during anesthesia were recorded from time of anesthesia administration, sleeping induction time, drug administration time and wake up time.

Bio-sample Collection
Urine Collection. From all the animals, 24 h urine was collected by placing individual animal in the 'metabolic cages', specifically designed for urine collection. These cages were designed in such a way that the animals have access for food and water, in addition the urine neither mixes with feces nor spillover water/feed. Urine collected was qualitatively analyzed using Ames Multistix strips (Siemens).
Blood Collection. The effect of test compound administration on various organ systems and metabolism was assessed by collection of blood and investigating the sensitive biomarkers. In view of this, blood was collected from all the animals through retro-orbital plexus using the heparinized capillaries. Briefly, animal was restrained, then the skin of neck gently scruffed, thus the eye made to bulge. Heparinsed capillary tube inserted medially and the blood was collected into tubes that were either pre-coated with anti-coagulants or plain tubes, as aliquots. Then an aliquot of blood samples were subjected for serum/plasma separation by centrifugation.
Vital organs/tissue collection. The process of vital organs/tissues that were collected was explained below.

Clinical laboratory investigations
Qualitative urine analysis. The urobilinogen, protein, pH, RBC, specific gravity, ketone, bilirubin and glucose were analyzed qualitatively in urine samples collected prior to vaccine administration and at different time points of post-exposure using Ames Multistix reagent strips (Siemens).
Clinical chemistry. Effect of vaccine administration on various organ systems were assessed by albumin, total protein, calcium and glucose levels along with liver function markers such as alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) levels in serum samples collected at different time points. In addition, the renal function markers viz., creatinine, total bilirubin and urea levels were also estimated in serum of the animals exposed with vaccine.
Hematology. The effect of test compound exposure on blood was investigated in terms of total white blood cell (WBC), red blood cell (RBC), hemoglobin (Hb), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, mean platelet volume (MPV) and differential leucocytes counts. These investigations were carried out in blood collected in to EDTA K2 vacutainer tubes at different time points.
Immunotoxicology. The immunotoxic effect of vaccine exposure if any, was assessed through tier-I and tier-II tests. The tier-I test includes a battery of investigations viz. CBC (Compete blood count), differential blood count, lymphoid organ weights, body weights, histopathology of lymphoid organs, spleen and bone marrow cellularity. When the tire-I tests yield evidence of immune dysfunction, then tire-II tests would be carried out. The tire-II tests include mitogen stimulation of B and T cells, primary (IgM, IgG) humoral response to T-dependent antigen, delayed type hypersensitivity response (DTH) and mouse ear swelling test (MEST).

Euthanization and necropsy
At the end of the experimental period the animals were fasted overnight (feed withdrawn and water provided) and blood was collected through retro-orbital plexus and euthanized either through cervical dislocation (mice and rat) or administration of high dose of anesthesia through marginal vein (rabbit). Then the animals were examined for lymphnode enlargement through gross examination process followed by necropsy and collection of vital organs as illustrated below.
Gross necropsy examination. The animals were thoroughly examined for any physical external abnormalities. Then the abdominal cavity and chest region opened and carefully observed for multitude of lesions if any, and any macroscopic changes in organs systems.
Collection of vital organs. The organs viz., brain, heart, lungs, liver, spleen, kidneys (left & right), gastrointestinal tract, pancreas, individual sex organs, thymus, thyroid, trachea, adrenals, bone marrow and site of injection were collected carefully and once again the individual organs were examined for gross changes. Then the weights of organs were recorded using sensitive balance, subsequently sliced adequately and transferred into 10% neutral buffered saline.
Histopathology. The effect of test compound exposure on various organs/tissues were assessed by 'histological examination'. The organs/tissues were processed by conventional methods after a minimum of 24 h fixation, subsequently paraffin blocks were prepared to obtain 4 μm sections. Then the sections were stained with Hematoxylin and Eosin and examined under a research microscope to unveil the cytological variations, if any. Histological changes in tissues were recorded and compared with corresponding controls.

Genotoxicity
The bone marrow micronucleus test was used to evaluate the genotoxic effect of vaccine exposure, if any. The femur bones of all the animals were collected and marrow was flushed using foetal calf serum. A fine colloid of the flushed out marrow was prepared and mounted on the slides after airdrying from the sediment after centrifuging at 800 rpm for 5 min. The staining was done using May-Gruenwald stain and followed by Geimsa stain. The scoring was done for 10 0 0 PCEs and the presence of NCEs and micro nucleated PCEs and the ratio (PCE/NCE) has been calculated.