Comparative assessment of humoral immune responses of aluminum hydroxide and oil-emulsion adjuvants in Influenza (H9N2) and Newcastle inactive vaccines to chickens

Abstract

Context Adjuvants are compounds used in the preparation of inactive vaccines to enhance the immune response. Aluminum hydroxide (alum) is one of the first compounds approved by the Food and Drug Administration, which is used as adjuvants in vaccine products for humans. Montanide ISA 70 is an oil-emulsion adjuvant and is used in poultry inactive vaccines. Objective In this study, the effects of alum adjuvant on the efficiency and induction of immune response in inactive vaccines of Influenza and Newcastle are compared with those of ISA 70. Materials and methods Six groups of 7-d-old specific-pathogen-free chickens were inoculated with 0.3 ml of the prepared vaccines via the subcutaneous route in the neck. Immune response in each group after 7, 14, 21, 31, 41, and 45 d was evaluated using the technique of hemagglutination inhibition. Results The results were compared using SPSS software. Results showed that vaccines containing adjuvant ISA 70 depicted a higher increase in the immune response and adjuvant of 20% alum is similar to adjuvant of ISA 70 in boosting the immune system. There was no statistically significant difference between 10% and 20% alum, but these adjuvants are visibly different from ISA 70. Conclusion In conclusion, alum can be used as an easily accessible, harmless, and effective adjuvant; however, to increase the immune period using the inactive vaccines for poultry, more research would be necessary.

Keywords:

• Adjuvant
• aluminum hydroxide
• humoral immune responses
• influenza (H9N2) and Newcastle vaccines
• montanide ISA70

Introduction

Influenza (AI) and Newcastle (ND) diseases are known to have a significant economic effect on the poultry industry (Arous et al. 2013, Brugh et al. 1979). Type A Influenza, subtype H9N2, belongs to the enveloped viruses of Orthomyxoviridae family with negative single-stranded and segmented RNA, which usually causes respiratory infections, decrease in egg production, and death in poultry (Lee et al. 2011).

Hemagglutinin (HA) and neuraminidase are important proteins on the envelope that play an important role in inducing a humoral immune response (Chauhan et al. 2013). ND virus is classified as Rubulavirus and belongs to the family Paramyxoviridae. The genome of this virus is a single-stranded, linear RNA with the size of 16–20 kb, and unlike that of Orthomyxoviruses, it is not a segmented genome (Alexander 2001).

The dangers of the infectious spread of the virus and its transmission to mammals, especially to humans, make it very important to render protection against these viruses (Alexander 2001). In Iran’s poultry industry, vaccination of birds is the most effective and the optimum method to fight against these infections.

Studies have shown that because of the side effects of using live vaccines, the inactive vaccines are a better alternative. In this regard and for improvement of the formulation and effectiveness of these vaccines, use of a proper adjuvant is necessary (Bowersock and Martin 1999).

Adjuvants are added to vaccines to increase the immune response to antigens. They can induce antibody production and potency, increase the duration of antibody response, and decrease the frequency of vaccination and the amount of necessary antigen in each dose of vaccine (Brito and O’Hagan 2014).

Nowadays, adjuvant in the treatment of diseases such as cancer are also used (Kazemi et al. 2015). Glenny et al. for the first time in 1926, showed the effect of adjuvants of aluminum compounds. Because these adjuvants are safe with no side effects, they were the first to be approved for use in human vaccinations in the United States. Today, these adjuvants are used in vaccine formulations (Arthanari et al. 2014). Aluminum salts (Alum) are the first and the most used adjuvants in vaccines, for more than 70 years (Lindblad 2004).

The first study using emulsion with Freund’s adjuvant go back to 1940 (Brito and O’Hagan 2014). This group containing a mixture of two immiscible materials is mixed together in the presence of one or several emulsifiers or surfactants. Emulsions that are used in vaccines can be categorized into two emulsions, water-in-oil (such as Montanide) or oil-in-water (such as MF59). Although water-in-oil emulsions are used for the long-lasting production of humoral immune response in the inactive poultry vaccines, they are not appropriate to be used in human vaccines, because they cause inflammation at the site of injection (Atmar and Keitel 2009).

In this study, adjuvants of alum with concentrations of 10% and 20% and ISA 70 were used to make inactive vaccines of AI and ND and were compared by using antibody measurements. With regard to sanctions on Iran and the problems in the import of montanide, we have to replace it with another material. Considering the price of alum, its easy availability, and its safety, it is suggested as an effective substitute for oil adjuvant of montanide.

Materials and methods

Antigens

AI viruses A/Chicken/Iran/101/1998/H9N2 and ND virus (V4) as antigen were donated by Razi Vaccine and Serum Research Institute.

Adjuvants

Oil adjuvant Montanide ISA 70 product of SEPPIC, Paris, France, was used to make the vaccines. Alum gel was donated by Razi Vaccine and Serum Research institute and levels of 10%, 20%, 30%, and 40% was used in vaccine preparation.

Chickens

In this study, 56 specific-pathogen-free (SPF) 1-week-old chickens were used. The eggs for these chickens were purchased from Venkeys (India) West Hatchery Company.

Preparation of vaccines

Oil adjuvant vaccines

To prepare the oil-in-water emulsion, a homogeny of oil adjuvant of Montanide ISA 70 with the inactive antigen of ND and AI with the ratio of 7:3 is used. The concentration of antigen had a minimum dosage of 10^8.5 in the aqueous phase (Pour et al. 2006).

Aluminum hydroxide gel vaccines

Before the use of alum gel as an adjuvant, first the absorbance capacity of the alum gel for viruses was measured. For this purpose, 10%, 20%, 30%, and 40% concentrations of alum gel in phosphate-buffered saline were prepared with the equal amount of antigen. Hence, after 4 h stirring, the solutions were centrifuged and the supernatants were surveyed by HA technique. All concentrations were of high absorption but in this study, the concentrations of 10% and 20% were used in the preparation of vaccines.

Hemagglutination assay (HA)

This technique has been explained by Masurel and his colleagues in 1981 (Rimmelzwaan et al. 1998).

Immunization of animals

In the laboratory, the 1-week-old SPF chickens were given 0.3 ml of the vaccines subcutaneously in the neck. Chickens were respectively separated in seven different vaccine groups (Table 1).

Table 1. Vaccination groups.

Groups	Type of vaccines
1	Newcastle with 10% aluminum hydroxide
2	Newcastle with 20% aluminum hydroxide
3	Newcastle with adjuvant of ISA 70
4	Influenza with 10% aluminum hydroxide
5	Influenza with 20% aluminum hydroxide
6	Influenza with ISA 70
7	Phosphate-buffered saline (PBS)

Blood sampling

Blood samples were taken in 7, 14, 21, 31, 41, and 45 d after vaccination. The blood samples were stored in an incubator (37 °C) for 1 h. Serum was separated and stored at 4 °C for HI antibody determination (Kallon et al. 2013).

Hemagglutination inhibition assay (HI)

Antibody titer was measured by the HI test. This test was performed as a standard method (Kallon et al. 2013).

Statistical analysis

To analyze the data of each group and compare them, Statistical Package for the Social Sciences software (SPSS Inc., Chicago, IL) was used. If P values are less than 0.05, then there is a significant difference between groups.

Results

Optimization of antigens absorption in the aluminum hydroxide gel

Concentrations of 10% and 20% of alum were used to prepare the vaccines. To optimize the time of antigen absorption, supernatants titer were investigated after 4 h by HA test. One hour after stirring, the HA titer of supernatants were zero. Hence, the best time of antigen absorption was 1 h after stirring of 10% and 20% gel for vaccine preparation.

Newcastle vaccines

Immune response of aluminum hydroxide gel vaccines

According to the numerical results, comparing the two concentrations of 10% and 20% adjuvant of alum in ND vaccines showed an increase in the immune response [in the test group] after 14 d of inoculation, and there was a sudden increase in the amount of antibody in the blood plasma. In the ND vaccine containing 10% of alum gel, increase in the immune response showed a steady trend and after 41 d of inoculation, this response showed a slight decrease.

In the case of ND vaccine containing 20% of alum, the antibody response was stable even after 21 d of inoculation and also until the last day of blood sampling. In addition, the immune responses at both these time points were slightly more than the immune response elicited by the vaccines containing 10% of alum gel.

Immune response of oil adjuvant vaccine

In the vaccines containing ISA 70 adjuvant, there was a significant increase in the immune response after second blood sampling (day 14 after inoculation). But, this increase was not steady and from day 31 after inoculation, the response decreased (Table 2).

Table 2. Immune response of SPF chickens to ND vaccine antibodies measured by HI.

Type of adjuvants	Day 7	Day 14	Day 21	Day 31	Day 41	Day 45
Aluminum hydroxide 10%	0	0	2.6	2.4	2.14	1.25
Aluminum hydroxide 20%	0	0	3.5	2.1	2.14	2.2
ISA 70	0	5	6.3	7	6.62	5.2

By comparing the results of adjuvants, on a diagram (Figure 1), it is noticeable that increase of immune response using oil adjuvants is more than the alum adjuvants, but from day 31 of vaccination on, the amount of antibody decreases. On the other hand, in the case of vaccines containing 20% alum adjuvant, immune response stays steady after day 31 of inoculation and until the last blood sampling, there is no decrease in the antibody.

Figure 1. Comparison of antibody titers (HI) measured of ND-vaccinated chickens.

Influenza vaccines

Immune response vaccines with adjuvant of aluminum hydroxide

According to the results in Table 3, the immune response for AI vaccines containing 10% adjuvant of alum in the second blood sampling (day 14 after inoculation) and in the third blood sampling (day 21 after inoculation) remained the same and in the last blood sampling after day 45, it decreased to zero.

Table 3. Immune response of SPF chickens to AI vaccine antibodies measured by HI.

Type of adjuvant	Day 7	Day 14	Day 21	Day 31	Day 41	Day 45
Aluminum hydroxide 10%	0	1	1	0.85	0.1	0
Aluminum hydroxide 20%	0	0.85	2.6	1.7	1.5	1
ISA 70	0	0.25	2.28	3.25	3.87	3.85

For AI vaccines containing 20% adjuvant of alum, the level of immune response increased significantly and reached maximum level by day 21 after inoculation, but it decreased after day 31.

Immune response with oil adjuvant vaccine

The AI vaccine containing ISA 70 adjuvant, after second blood sampling (day 14 after inoculation), showed a significant increase in immune response, which remained high until the last blood sampling. Comparison of the results in the figure shows that increase of immune response for ISA 70 adjuvant is similar to that by alum adjuvant of 20% until day 21 after inoculation, but after day 21, the decrease in the antibody was faster for 20% alum adjuvant. Also, the decrease in the antibody for 10% alum adjuvant is more rapid in comparison with 20% alum adjuvant (Figure 2).

Figure 2. Comparison of antibody titers (HI) measured of AI-vaccinated chickens.

Discussion

AI and ND diseases are very important and every year, they cause heavy financial loss in the poultry industry. To prevent these diseases, it is important to produce inexpensive vaccines.

It has been more than 70 years that adjuvants are being used in vaccines to increase the immune response in host subjects. Adjuvants are additives that increase the antigen’s ability to induce an immune response (Stils 2005). In general, one of the important roles of adjuvants is to reduce the necessary amount of antigen in each vaccine dose (Luke and Subbarao 2006).

Vogel and Powell had analyzed more than 100 adjuvants in the year 1995 (Arous et al. 2013). Because most of these adjuvants are hard to produce, expensive, and have poisonous effects, their usage is limited, which were explained in the year 2000 by Stewart-Tull (Atmar and Keitel 2009).

Alum is one of the first and oldest adjuvants, which is used in producing vaccines for humans and is approved by the Food and Drug Administration (FDA) (Arous et al. 2013, Arthanari et al. 2014, Luke and Subbarao 2006, Vasievich and Huang 2011). In the year 2000, Gupta and Rost reported that aluminum compounds, especially aluminum phosphate and alum, were used as adjuvants in the production of human vaccines. Adjuvants of aluminum, unlike emulsions, interact with antigens using electrostatic forces in the aqueous and non-aqueous phases. They also interact with antigens by hydrophobic reactions, van der Waals forces, and hydrogen bonds (Stils 2005). Another report said alum may not be able to effectively induce the immune system (Arthanari et al. 2014).

Mishra et al. investigated that alum-HBsAg formulation induced humoral immunity significantly because the IgG1 was increased in serum (Mishra et al. 2014).

In the year 1962, Perez-Rebelo used alum in their study on ND virus. In 1980, Larghi and Nebel used a concentration of 50% alum gel to absorb Rabies virus. Reid and Blackall in 1987 suggested the use of 25% alum gel for Haemophilus paragallinarum (Gupta et al. 1995, Perez-Rebelo 1962, Reid and Blackall 1987).

In the present study, for antigen absorption of alum, one should bear in mind that all samples of 10%, 20%, 30%, and 40% of alum showed complete absorption in the first hour. Based on the results of this study and previous studies and to reduce the chance of tissue damage at the site of injection, the concentrations of 10% and 20% alum gel were selected for use (Stils 2005).

A significant effect of antigen depot was explained by Herbert (1967). The amount of antibody after inoculation of antigen in an emulsion of water-in-oil was compared by him with a daily injection of antigen in saline for over a 1-month period. Although in both groups the amount of antibody was high, in the case of injection of the emulsion, the amount of antibody remained high, unlike the saline group, in which the antibody decreased after the daily injections were stopped (n = 14).

It is said that antigen exists at the site of inoculation up to 7 weeks after the injection. This opinion was experimented by Harrison in the year 1935. Considering that all vaccines in this study contained the same dose of vaccines, which means that they had the same amount of antigen, a significant difference in the antibody response for the two vaccinated groups of 10% and 20% alum is visible. It means that increase in the amount of alum increases the immune response. Therefore, according to previous studies and the results of this study, we can assume that the performance of vaccines containing 20% adjuvant of alum is better than the ones containing 10% of alum. This higher performance is based on the increase in the immune response related to a longer period of antigen depot.

Stone et al. compared the immunity of three commercial oil-based vaccines with one vaccine prepared using alum against the ND poultry disease. The immunity produced by oil-based vaccines, in all cases, was significantly higher than that induced by the vaccine containing alum (Stone et al. 1980). In addition, in the year 2011, it was reported that oil adjuvants result in better performance compared with alum (Liu et al. 2011).

Box and Furminger reported that the HI titer of ND inactivated vaccines containing alum resulted in the maximum in the 4th week after vaccination. Then, the titer was decreased, insofar decline to minimum titer in 12th week after vaccination (Box and Furminger 1975).

ND disease vaccines in this study produced consistent results with the concentration of 10% serum-grade alum at 21 d after vaccination (3 weeks), but at day 45 after vaccination, the immune response slightly reduced.

The ND vaccine with 20% of alum gel adjuvant produced antibody titers that were constant from 3 weeks to 45 d after inoculation. Yamanaka in 1993 reported that 2 weeks after vaccination with inactivated ND vaccine containing montanide produced an immune response that is more than eight times the response elicited by the inactivated ND disease vaccine at the same time period. Stone et al. (1980) also reported such consistent results (Yamanaka et al. 1993).

In the present study, oil-emulsion of ND vaccine enhanced the immune response in 14 d after inoculation. This response was decreased in 45 d after inoculation. The results show that immune response stimulation rate of oil vaccine of ND disease is more rapid compared with alum gel. However, the titers decreased in both groups at day 45.

Use of the different volume of adjuvant is the reason for this difference. In other words, the alum vaccines containing up to 20% of adjuvant in a vicinal dose, whereas, in oil-emulsion vaccines, the oil-emulsion volume could be up to 70%. Perhaps, one of the reasons for the effectiveness of oil vaccines to stimulate and durability of immune responses is the use of the higher volume of adjuvant in the vaccine combination.

The AI vaccine adjuvant alum (10%) increased the antibody titer at 14 d after inoculation and remained stable until 31 d after inoculation. The flu vaccine adjuvant alum (20%) showed antibody titer at 21 d after inoculation and reached its maximum level. Immune response of AI oil vaccine continued to increase between 14 and 45 d.

According to results, both vaccines simultaneously increased antibody titers but the persistence of titers in the group what vaccinated with AI alum vaccine was lower than oil vaccine. It is another reason for the superiority of oil vaccines.

Statistical analysis of the results showed no significant difference in AI and ND disease vaccine adjuvants alum, 10%, 20%, and ISA 70. However, there was a statistically significant difference between alum and montanide, but as the results show, alum is also able to induce a high antibody response.

The AI vaccine for ND disease vaccine showed similar results, the difference being a weaker immune response to ND vaccine, which is likely due to the lower quality of the AI antigen. The stability of the immune response to AI vaccine oil was seen in all groups. Hence, given the vaccine immune response and the increased longevity is concerned, this can replace the ISA 70 adjuvant alum for use in certain critical times.

A vaccination plan was employed in poultry flocks according to a study conducted in 2011 by Lee et al. It was shown that two-dose vaccination program with H9N2 vaccine with alum as adjuvant and adjuvant oil could enhance the immune response, compared with the vaccination program with one dose of vaccination, thus improving the immune response (Lee et al. 2011).

This strategy can greatly protect against bird flu in the poultry industry and is also economically effective. Thus, it can be suggested that alum adjuvant and inactivated adjuvant vaccine oil can be used together in poultry.

It can be due to the high price of oil in the adjuvant alum gel vaccine emulsion and low price, it is recommended gel use of vaccines in vaccination programs and to earn higher grades, the percentage increased gel or a booster dose was administered.

Due to the similarity of the numerical results obtained with 20% alum adjuvant and ISA 70 to increase immune, it is suggested that the amount of aluminum in the vaccine formulation is used more than 20%.

Considering that 70% of oil vaccines is adjuvant, so to reduce the dose of 0.3 to 0.1 in under a week-old chickens, suggested ISA 70 replaced that alum. ISA 70 that used in poultry vaccines is an expensive, rare, and imported raw material in Iran. In addition, hi-tech equipment was needed to homogenize it. Therefore, according to the results and with regard to the sanctions against Iran, replacing the adjuvant alum gel adjuvant with cheaper, acceptably efficient, accessible, and non-risk adjuvants (approved by the FDA) is essential.

Conclusion

With regard to sanctions on Iran and the lack of import montanide, alum can be used as an easily accessible, harmless, and effective adjuvant substitute to ISA 70; however, to increase the immune period using the inactive vaccines for poultry, more research would be necessary.

Declaration of interest

This study has been supported by a grant provided from Razi Vaccine and Serum Research Institute, Karaj, Iran. The authors report no declarations of interest.

Acknowledgements

The authors express their gratitude to Dr. Alireza Bahonar and Mahnaz Haddadi who supported the development of this work.