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Acta toxicológica argentina

versión On-line ISSN 1851-3743

Acta toxicol. argent. vol.27 no.3 Ciudad Autónoma de Buenos Aires dic. 2019

 

ARTÍCULO ORIGINAL

Reactogenicity of Bordetella pertussis vaccines formulations using aluminum adjuvants in Sprague Dawley rats

Reactogenicidad de formulaciones de vacunas de Bordetella pertussis utilizando adyuvantes de aluminio en ratas Sprague Dawley

 

Oliva Hernández, Reynaldo*; Fariñas Medina, Mildrey; Hernández Salazar, Tamara; Hernández Cedeño, Mabel; Acevedo Grogues, Reinaldo; García Rivera, Dagmar

Department Head of Animals Models and Experimental Toxicology. Finlay Institute of Vaccine, P.O. Box 16017, La Lisa, Havana, Cuba.

*roh@finlay.edu.cu; reyolivacuba@gmail.com

Recibido: 6 de mayo de 2019
Aceptado: 22 de noviembre de 2019


Abstract

Combined vaccines for childhood are a strategy in the prevention of several diseases. These can maximize protection and decrease immunization schedules in children. New candidates are getting closer to being able to meet these needs, but they raise numerous strategic questions related to formulation and regulatory aspects. In addition to being immunogenic and protective must have low reactogenicity when combined with other antigens. Adjuvants are important components in achieving these combinations. Therefore, a reactogenicity study was designed for two Bordetella pertussis formulations containing hydroxide or aluminum phosphate in Sprague Dawley rats. Both formulations dose were administered in 0.2 mL intramuscularly. Clinical evaluations, body weight, water consumption, food, temperature, muscle volume, dermal irritability and pathological studies with special interest at the inoculation site were carried out. Only differences in body temperature and muscle volume were found with a slight increase in values with return to normal. The macroscopic study showed lesions at the site of inoculation, consid­ered characteristics of aluminum adjuvants, such as granulomatous abscesses and the increase in regional lymph nodes near the inoculation site. As conclusion, there are no differences between the formulations of B. pertussis with hydroxide or aluminum phosphate resulted in low reactogenicity.

Keywords: Reactogenicity; B. pertussis; Adjuvants; Sprague Dawley rats.

Resumen

Las vacunas combinadas resultan una estrategia importante en la obtención de vacunas múltiples para la infancia y el uso de adyuvantes es un componente de gran valor en lograr estas combinaciones, además de ser inmunogénicas y protectoras deben tener baja reactogenicidad, cuando se combinan con diferentes antígenos. Por esta razón, se diseñó un estudio de reactogenicidad a dos formulaciones que contenían hidróxido y fosfato de aluminio con antígenos de Bordetella pertussis en ratas Sprague Dawley. Se administró a cada grupo de ensayo una dosis correspondiente de ambas formulaciones en 0,2 mL por vía intramuscular. Se realizaron observaciones clínicas, comportamiento del peso corporal, consumo de agua, alimentos, temperatura corporal, volumen muscular, irritabilidad dérmica y estudios anatomopatológicos macroscópicos, con especial interés en el sitio de inoculación. No se observaron síntomas, ni muertes en los animales durante el estudio. Tampoco se encontraron diferencias entre los grupos experimentales en cuanto al peso corporal, el consumo de agua y de alimentos; los estudios de temperatura corporal y volumetría muscular evidenciaron un ligero incremento en los valores, los cuales involucionaron rápidamente a la normalidad. En el estudio anatomopatológico macroscópico se observaron lesiones a nivel del punto de inoculación, consideradas propias de los adyuvantes que contienen aluminio, tales como formaciones abscedadas de tipo granulomatosas y el aumento de los ganglios linfáticos regionales cercanos al punto de inoculación. Se concluye que las formulaciones en hidróxido y fosfato de aluminio con antígenos de B.pertussis resultaron ser de baja reactogenicidad.

Palabras clave: Reactogenicidad; B.pertussis; Adyuvantes; Ratas Sprague Dawley.


Introduction

Obtaining multiple or combined vaccines has been a justified strategy, in addition to the possibility of reducing the number of immunizations to which children are subjected, mainly for the prevention of several diseases caused by different strains or serotypes of the same microorganism (FDA 1997; Walker and Dull 2017). There are several combined vaccines designed for the prevention of diseases such as Diphtheria, Tetanus and Pertus­sis (DPT vaccine), pentavalent (HeberPenta®-L), against Haemophilus type b, Hepatitis B plus DPT, Shigella combined vaccine, against malaria and cancer, among others; this vaccines can have in their formulations new or known adjuvants (Ba­cardí et al. 2013; Silva et al. 2015; Pandey et al. 2016; Rampling et al. 2016).

Finlay Institute of Vaccines works in obtaining vaccines for the prevention of different diseases and one of its lines is combined vaccines for childhood. In this sense, B. pertussis (Bp) antigens are currently evaluated as one of the components of different combined vaccines. The component of inactivated cells of Bp in the current DPT and HeberPenta®-L vaccines of the Cuban immunization scheme, are adjuvanted in aluminum hydroxide (Al(OH)3) and aluminum phosphate (AlPO4) as well, similar to others kind of vaccines like VA-MENGOC-BC® as well mentioned before (Oliva et al. 2019). One of the critical elements for multivalent vaccines containing inactivated Bp cells is reactogenicity or local response at the immunization site (FDA 1997). Considering the possibility of combining Bp cells with other antigens that need to be adjuvanted in AlPO4, a reactogenicity study was designed two formulations with this antigen adjuvanted to Al (OH)3 or AlPO4 in Sprague Dawley (SD) rats.

Materials and methods

Animals and husbandry

Male SD rats were purchased from CENPALAB, Havana Cuba (from Spanish: Centro Nacional para la Producción de Animales de Laboratorio) at an age of 7-8 weeks and were housed in Tecniplast® rat cages at the Animal Care Facility at Finlay Institute of Vaccine. Dimensions and model: 1354G Eurostandard Type IV, 595 x 380 x 200 mm, floor area 1820 cm, PEI plastic and BPA-Free. Rats were provided with specialized feed for rodents (ALYco®), and the water used was provided in acidified (2.5-2.7 pH) water bottles (750 mL volume). Both food and water 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 commencement 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.

Formulation and vaccination protocol

The formulations were prepared (Table 1) from a batch of Bp (strains 165, 509 and 134) obtained in the production plant of the Finlay Institute of Vaccines. The adsorption process used for the combination of Bp (whole-cell inactivated) and Al(OH)3 was similar to DPT vaccine. The Bp was absorbed in AlPO4 with pH from 6.0 to 6.5 at room temperature, in presence of 0.9% NaCl as a buffer solution. Both formulations Bp were maintained at a concentration of 32 OU/mL, while concentration of aluminum (Al+) was 0.7 mg/mL approximately for each adjuvant.

Table 1. Experimental design

Study lasted 7 days; for which three treatment groups by randomization were formed, receiving a dose of the formulated vaccines under study (Table 1). Rats were immunized intramuscularly (IM) to use the same route used in humans, in the posterior middle region of the inner side of both legs (100 μL in each leg). This study was designed following the recommendations and guidelines issued by the WHO for the evaluation of vaccines (WHO 2013).

Clinical observations and symptoms

All observations started from the experimental zero time (T0), which was considered as the same day of vaccination. Animals were monitored daily (three times a day during first 72 hours) paying special attention to the rats when evaluating their food and water intake, body weight, diameter of the leg muscles, skin and temperature.

Body weight, water and food consumption

At the time of inoculation, rats were weighed to determine their starting weight at the beginning of the study. All animals were identified by the ear punch-out method and weighed at week (end-point) to monitor their weight as a measure of toxicity.

Water and food consumption were measured at the start of the vaccination protocol (T0) and alternating 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 rats was measured with a mercury clinical thermometer thin (Hemeco, China) rectally. Body temperature was measured before the inoculation and 72 hours after (intervals of time of 4, 24, 48 and 72 hours).

The evaluation of the muscle diameter was per­formed as previously described for rats and mice (Oliva et al. 2019; Fraleigh et al. 2019) with a digital caliper (Mastercraft, Electronic Caliper with digital display, 6’’,150 mm) by measuring the diameter of the inoculated limb before the intramuscular vaccination at 4, 24, 48 and 72 hours after vaccination to assess local inflammation.

Dermal irritability

Dermal irritability test (DI) was performed in similar way and as previously described for rats (Oliva et al. 2019). The test is made by Draize method modified and as predictive element of local reactogenicity for clinical essays (Draize et al. 1944) and has been recommend by World Health Organization (WHO 2013). Score of DI was evaluated in same times previously described for temperature and muscle diameter.

Anatomopathological studies

The anatomopathological studies for gross necropsy were performed immediately after euthanasia at 7 days. All organs and sites (lymph nodes) of vaccine administration were examined macroscopically.

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 male SD rats increased their body weight during the 7 days of the study, no statistically significant difference was observed between groups (Figure 1). The weight increase curves of the rats were similar to those observed for this specie and in line with the growth curves available from Charles River and similar to other studies made by us with historical data from our animal’s facility as well.


Figure 1.
Body weight time course of rats with formulations adjuvants B. pertussis vaccine. Each value represents the average ± SEM of the 10 animals in the groups.

We also evaluated the amount of water (a general average of 66.7 mL, Figure 2) and food (a general average of 29.1 g, Figure 3) consumed by the SD rats over the span of the experiment design, which showed not significant difference between any of the groups measured in the study. Results correspond to the historical values observed for rats of this category in our facilities (Sosa et al. 2005; Fariñas et al. 2014; Oliva et al. 2019).


Figure 2.
Water consumption time course ofrats with formulations adjuvants B. pertussis vaccine. Each value represents the average ± SEM of the 10 animals in the groups.


Figure 3.
Food consumption time course of rats with formulations adjuvants B. pertussis vaccine. Each value represents the average ± SEM of the 10 animals in the groups.

The body temperature recorded during the different evaluations carried out post-inoculation did not show significant differences between groups of treatments (Figure 4), it behaved within the physiological ranges reported for the species (CCAC 1984). In order to assess the inflammation induced by the formulations adjuvanted vaccines Bp, administered intramuscularly as element of reactogenicity and local response, the muscle diameter of the legs was measured before and after receiving the IM vaccination. We found significant differences between controls and treated with formulations of AlPO4 and AL(OH)3 with Bp antigens at 4 and 24 hours post-inoculation and not between the formulations (Figure 5).


Figure 4.
Corporal temperature time course of rats with formulations adjuvants B. pertussis vaccine. Each value represents the average ± SEM of the 10 animals in the groups.


Figure 5.
Muscle diameter time course of rats with formulations adjuvants B. pertussis vaccine. Each value represents the average ± SEM of the 10 animals in the groups. Asterisk (*) indicates significant difference from the rest of the groups (p < 0.05).

The dermal irritability test performed by the Draize method allowed discarding through the skin readings at the site of administration of the two formulations; the absence of erythema, edemas, eschar or papules, being the scale of dermal irritation for both 0,0.

In general, macroscopic studies performed on all organs and systems for each of the SD rats studied did not show any lesions suggesting acute toxicity. At the site of inoculation, abscessed white-grayish formations were observed in the animals receiving the formulations. It may be related to the adjuvants AlPO4 and AL(OH)3, as this is a reported characteristic of them (Sosa et al. 2005; Oliva et al. 2019). Also, the deep popliteal and inguinal lymph nodes were seen size increase.

Discussion

From the past century, aluminum in different form has been used to improve the immunogenicity of vaccines and it’s the most studied and used in combined vaccines as adjuvant and current evidence supports the safety of aluminum ad­juvants in vaccines (Eickhoff and Myers 2002; CDC 2019; DeStefano et al. 2019). Particularly, Bp vaccines have a certain reactogenicity attribu­tion, especially those of whole cell (WHO 2015). However, when Bp are combined with other Ag, be of whole cell or acellular, his reactogenicity could be decrease or be diluted with other Ag present in formulation, unlike when they are the only ones present. For other side, aluminum as component and adjuvant for vaccines continues to be recognized by experts and the World Health Organization of low risk (WHO 2012; CDC 2019) and they are important as strategic part of the news formulations of combined vaccine.

The local response or reactogenicity of vaccines in clinical trial is a critical element, more for combined vaccines, because can have many different Ag in one formulation. Reason why preclinical toxicity essays become important and with highs predictive value to human test. In addition, in these preclinical essays, also taken in account systemic effects, like body weight, fever, and others.

Effects on animal body weight usually manifest in either a decrease in gain compared to controls and placebo or absolute losses of body weight, and are dependent on the toxicity of the product tested. This is a determinant parameter in toxicological studies. The evaluation of body temperature, local inflammation and redness of the skin at the injection site for the IM route can be related to some kind of systemic effect and they are parameters considered predictive for clinical trials (Oliva et al. 2019); where observations is based in expected and unexpected adverse events, which include fever, headache, pain at the site of administration, redness, abdominal pain, vomiting, diarrhea, among others (Ochoa et al. 2006; García et al. 2011).

In our case, both adjuvant formulations (AlPO4 and Al(OH)3) Bp vaccine can be considered of low reactogenicity similar to current combined vaccines with Bp as component Ag. Because, when a fever (>38°C) is induced by some vaccines by intramuscular injection it is registered within the first 24 hours (Hamilton 2013). We measured the temperature of the rats before and after vaccination during 72 hours to determine whether fever was induced. While a traditional fever was never induced in our study, generally in combined vaccines this can be seen and is an element to keep in mind.

The increase in muscle volume that implies difference between controls and vaccinates is explained by the inflammatory process caused by hydroxide and aluminum phosphate used as adjuvants (Sosa et al. 2005; Fariñas et al. 2014; Oliva et al. 2019), and by the Bp itself, which due to its inactivated cell nature generates a local inflammatory response. This reaction lasted approximately 24 hours, since at 48 hours there were no differences between controls and treated. However, the absence of symptoms of lameness, pain or some type of locomotion difficulty evidence for both adjuvants Bp, suggest that vaccine formulations are tolerable and support more the evidences on aluminum safe (DeStefano et al. 2019). Moreover, Draize method scale through skin lectured, clas­sify these formulations as potentially non-irritating to the skin, according to the criteria established in the literature (Draize 1944; OECD 2004). Other similar studies made for us, show usefulness of this method (Oliva et al. 2019) and comply with the OECD guidelines (OECD 2004).

The fact that male SD rats did not experience any adverse side effects nor where there changes in any of the organs evaluated in gross necropsy, would suggest that the formulations does not toxic. Nevertheless, on the local and inflammatory response of the lymphatic organs (popliteal and inguinal lymph nodes) and immune system were activated, showing an increase similar to other vaccines, being recognized as a normal immunological event with a direct related to the adjuvant and Ag (Sosa et al. 2005; Ahrendt et al. 2008; Sainte-Marie 2010; Bacardí et al. 2013; Oliva et al. 2019; Tamargo et al. 2019).

Together, the data suggests that not only low reac­togenicity of vaccines formulations of inactivated Bp cells adjuvanted in phosphate or aluminum hydroxide were similar, but it is also potentially non-toxic in a male SD rat model. Also, suggest that this antigenic component can be used in combined vaccines with either adjuvant, not implying this any change in their reactogenicity.

Acknowledgments:

This work was supported by Finlay Institute of Vaccine, Havana, Cuba (IFV). The authors are also thankful for the technical assistance by Maria Onelia González Socarras, Alex Quintero Perez, Darcy Nuñez, Yolanda Valdes, Aylín Amador and to all people from animal facility.

References

1. Ahrendt M., Hammerschmidt S.I., Pabst O., Pabst R., Bode U. Stromal cells confer lymph node-specific properties by shaping a unique microenvironment influencing local immune responses. J Immunol. 2008;181(3):1898-1907.         [ Links ]

2. Bacardí D., Cosme K., Aldana L., Merino N., Suárez J., Mosqueda O., Urquiza D., Romero J., Madrigal R., Amaya R., Hernández L. Preclinical safety testing of the Quimi-Hib® vaccine adjuvanted with aluminum phosphate during product development. Biotecnología Aplicada. 2013;30(2):118-124.         [ Links ]

3. Canadian Council on Animal Care (CCAC). Physiologycal & Nutritional Parameters. In: Guide to the care and use of experimental animals. CCAC, Ottawa, Canada. 1984; Vol. 1, p. 83.         [ Links ]

4. Centers for Disease Control and Prevention (CDC). Vaccines and Preventable Diseases. Types and Composition of Diphtheria, Tetanus, and Pertussis Vaccines. [accessed 8 March 2019] .Available at: https://www.cdc.gov/vaccines/vpd/dtap-tdap-td/hcp/about-vaccine.html.         [ Links ]

5. DeStefano F., Bodenstab H.M., Offit P.A. Principal Controversies in Vaccine Safety in the United States. Clinical Infectious Diseases. 2019;69(4):726–731.

6. Draize J.H., Woodard G., Calvery H.O. Methods for the Study of Irritation and Toxicity of Substances Applied Topically to the Skin and Mucous Membranes. Journal of Pharmacology and Experimental Therapeutics. 1944;3(82):377-90.         [ Links ]

7. Eickhoff T.C., Myers M. Workshop summary Alu­minum in vaccines. Vaccine. 2002;20(Suppl 3):1–4.

8. Fariñas M., Oliva R., Infante J.F., Valdez Y., Nuñez D., Valmaceda T., Hernández T., Fernández S. Ensayo piloto de inmunogenicidad y toxicidad preclínica de la vacuna Salmonella typhi conjugada en ratas Sprague Dawley. Retel 2014. [accessed 20 March 2019]. Available at: http://www.sertox.com.ar/modules.php?name=Content&pa=showpage&pid=936        [ Links ]

9. Food and Drug Administration (FDA). Guidance for Industry for the Evaluation of Combination Vaccines for Preventable Diseases: Production, Testing and Clinical Studies Docket No. 97N-0029. 1997. [accessed 21 June 2018]. Available at: http://www.fda.gov/cber/cberftp.html.         [ Links ]

10. Fraleigh N.L., Oliva R., Lewicky J.D., Martel A.L., Acevedo R., Dagmar G.R., Le H.-T. Assessing the immunogenicity and toxicity of the AFPL1-conjugate nicotine vaccine using heterologous and homologous vaccination routes. PLOS ONE. 2019;14(8):e0221708.         [ Links ]

11. García H.M., Thompson R., Valera R., Fando R., Fumane J., Jani I., Mirabal M., Armesto M.I., Songane M.L., Sonia N., Celeste A.M., Viegas J., Samo S., Melembe E., Bila A., Cemá D., Mabumo C.C., García L., Cedré B., Año G., Martínez J.C., Mandarioti A., Lugones J., González D., Baró M., Hernández J., Talavera A., Solis R.L., Sierra G., Barberá R., Domínguez F., Gutiérrez C., Campa C., Garrido I., Menéndez J. A single dose of live-attenuated 638 Vibrio cholerae oral vaccine is safe and immunogenic in adult volunteers in Mozam­bique. VacciMonitor. 2011;20(3):1-8.         [ Links ]

12. Hamilton J.L. Evaluation of fever in infants and young children. Am Fam Physician. 2013; 87(4): 254-260.         [ Links ]

13. Ochoa R.F., Baró I.M., Menéndez J., Triana T., Mi­rabal M., Armesto M., Rodríguez V., Valmaseda T., Fernández Y., Aranguren Y., Creadh L., Domínguez R., Bayolo J.V., Alvarez M., Peña M.A., Rodríguez M.C., Solís R.L., Domínguez F. Reactogenicidad e inmunogenicidad de una nueva vacuna de tox­oide tetánico y diftérico con concentración reducida en adolescentes cubanos. VacciMonitor. 2006;15(2):13-17.         [ Links ]

14. Oliva R., Fariñas M., Infante J.F., Hernández T., Nuñez D., Quintero A., Campa C., Sierra G. Local tolerance study of the VA-MENGOC-BC® anti­meningococcal vaccine in Sprague Dawley rats. Evaluation at 24 and 36 months of shelf. Vacci­Monitor. 2019;28(1):9-18.         [ Links ]

15. Organization for Economic Cooperation and De­velopment (OECD). Guideline for the Testing of Chemicals No 404. Paris: OECD; 2004. [Accessed 20 March 2019]. Available at: http://www.oecd.org/env/testguidelines.         [ Links ]

16. Pandey M., Mortensen R., Calcutt A., Powell J.,Batzloff M.R., Dietrich J., Good M.F. Combina­torial Synthetic Peptide Vaccine Strategy Protects against Hypervirulent CovR/S Mutant Streptococci. J Immunol. 2016;196(8):3364-3374.         [ Links ]

17. Rampling T., Ewer K.J., Bowyer G., Bliss C.M., Edwards N.J., Wright D., Payne R., Venkatraman N., de Barra E., Snudden C.M., Poulton I.D, de Graaf H., Sukhtankar P., Roberts R., Ivinson K., Weltzin R., Rajkumar B., Wille-Reece U., Lee C., Ockenhouse C., Sinden R.E., Gerry S., Lawrie A.M., Vekemans J., Morelle D., Lievens M., Ballou R.W., Cooke G.S, Faust S.N., Gilbert S., Hill A.V.S. Safety and High Level Efficacy of the Combina­ tion Malaria Vaccine Regimen of RTS,S/AS01B With Chimpanzee Adenovirus 63 and Modified Vaccinia Ankara Vectored Vaccines Expressing ME-TRAP. The Journal of Infectious Diseases. 2016; 214(5):772–781.

18. Sainte-Marie G. The lymph node revisited: de­velopment, morphology, functioning, and role in triggering primary immune responses. Anat Rec. (Hoboken). 2010;293(2):320–337.

19. Silva A., Mount A., Krstevska K., Pejoski D., Hardy M.P., Owczarek, C., Scotney P., Maraskovsky E., Baz Morelli A. The Combination of ISCOMATRIX Adjuvant and TLR Agonists Induces Regression of Established Solid Tumors In Vivo. J Immunol. 2015;194(5):2199-2207.         [ Links ]

20. Sosa E., Sifontes S., Infante J.F., Díaz D., López Y., Pérez V., Hernández T., Riverón L., Valdés Y., García I., Fariñas M., Parajón E., Rodríguez N. Estudio de tolerancia local de la vacuna vax-TyVi® en ratas Sprague Dawley. VacciMonitor. 2005;14(1):21-27.         [ Links ]

21. Tamargo Santos B., Bungau S., Fleitas Perez C., Marquez Napoles Y., Infante Bourzac J.F., Oliva Hernández R., Ramírez González W., Cioca G., Aleya L., Sierra González V.G. Immuno-toxico­logical Evaluation of the Adjuvant Formulations for Experimental Anti-meningococcal Vaccines without Aluminum Hydroxide. REV.CHIM (Bucha­rest). 2019;70(4):1251-1257.         [ Links ]

22. Walker R., Dull P. Combination vaccine strategies to prevent enteric infections. Vaccine. 2017;35(49, Part A): 6790-6792.         [ Links ]

23. World Health Organization (WHO). Global Advisory Committee on Vaccine Safety, June 2012. Weekly Epidemiol Rec. 2012; 87(30):281–287.

24. World Health Organization (WHO). Guidelines on the evaluation of vaccine adjuvants and adjuvanted vaccine, Technical Report Series No 987, Annex 2. Geneva: WHO; 2013. [Accessed 13 March 2019]. Available at: http://www.who.int/biologicals/vaccines/nonclinial_evaluation_of_vaccines/en/         [ Links ]

25. World Health Organization (WHO). Pertussis. 21 May 2015. [Accessed 8 March 2019]. Available at: https://www.who.int/biologicals/vaccines/pertussis/en/.         [ Links ]

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