Serological baseline, antibody stability and efficacy of different types of avian influenza (H5) vaccines

Abstract

Importance

Evaluating Iran’s national highly pathogenic avian influenza (HPAI) control program can inform vaccine selection, optimize immunization duration, guide exit strategies, and assess hemagglutination inhibition (HI) and serum neutralization (SN) methods.

Objective

To establish a serological baseline, assess antibody stability, and compare the efficacy of three HPAI (H5) vaccines.

Methods

We analyzed over 9,000 blood samples and 6,420 swabs from approximately 1.5 million birds up to 64 weeks old. HI (β, α), RT-PCR, and SN tests were conducted, with statistical analysis performed using two-way ANOVA.

Results

The serological baseline (GM titer) using H5N8 antigens from A/Chicken/Iran/162/2016 varied. The Re6+Re8 vaccine produced higher and more stable HI β titers than the H5N3 and baculovirus vaccines. Serum HI α neutralization ability was similar for Re6+Re8 and H5N3 vaccines, both 100 times greater than the baculovirus vaccine. Neutralization indices for H5N3, Re6+Re8, and baculovirus vaccines were 4.7, 4.5, and 4.2 (log2), respectively.

Conclusions and Relevance

After two vaccinations, Re6+Re8 exhibited the most stable HI β antibody response, while H5N3 had the highest neutralization index, surpassing Re6+Re8 by 0.2 and the baculovirus vaccine by 0.5. These findings highlight discrepancies between HI β and SN test results, with SN being a stronger indicator of protective titers due to its in vivo methodology, compared to the in vitro HI assay.

INTRODUCTION

Influenza is a highly contagious zoonotic disease, with wild aquatic immigrant birds as its reservoir. The cause of this disease is a virus belonging to the Orthomyxoviridae family with the genus Alphainfluenzavirus. Influenza viruses are classified into four main types: A, B, C, and D, with type A being pathogenic for humans and birds [1,2]. The difference in the surface epitope of hemagglutinin (HA) and neuraminidase (NA) determines the phylogenetic division into 16 HA (H1-H16) and 9 NA (N1-N9) subtypes. New subtypes H17, H18, N10, and N11 have been identified from bats in Guatemala. H5N1 highly pathogenic avian influenza (HPAI) viruses (lineage A/goose/Guangdong/1/1996) were first identified in China and spread to the Hong Kong bird market. Multiple subclades recombined with other avian influenza virus subtypes, causing the emergence of newer H5Nx subtypes, including H5N8, H5N6, H5N5, H5N3, H5N2, and H5N9.

Subtypes H5N8 and H5N6 from a new clade 2.3.4.4 (originated from clade 2.3.4.6) were noticed. Clade 2.3.4.4 was able to cause a widespread incidence with high mortality birds due to its rapid transformation and global spread [3,4]. The first official report of the H5N1 subtype in Iran was in 2006, and the first identification of the influenza disease under the H5N8 type (clade 2.3.4.4) was in November 2016 [5].

The irrational use of antiviral agents may confer the mutation in challenging viruses, most notably the avian influenza virus [6]. Some countries, including Iran, have started using inactivated HPAI vaccines in their control programs to further reduce economic damage. Several vaccine manufacturing technologies have been developed [7]. Complete virus vaccines inactivated with chemical agents and oil emulsions are among the most widely used vaccines for influenza and other viral diseases, e.g., Infectious bursal disease and Newcastle disease virus [8,9]. The effect of probiotic use in poultry feed could decrease vaccine failures and boost the immune parameters as well [10]. Recombinant live vaccines have been approved for use in several countries. In the national vaccination programs worldwide, the highest consumption was for inactivated oil vaccines (about 95.5%), with only 4.5% of the vaccines used belonging to the group of recombinant vaccines [11].

Experimental studies have shown that different vaccines can provide adequate clinical protection against the challenge of influenza disease. Still, it is crucial to verify whether these findings could be generalized to farm conditions, such as measuring maternal antibodies to determine the suitable age for vaccination [3,12,13]. The need for sampling and tests regarding serum changes and virus identification poses a significant challenge for many laboratories in developing countries [14,15]. Following the H5N8 avian influenza outbreak in the country in 2016–2017 and the heavy economic losses caused by the removal and destruction of more than 25 million commercial layers, Iran’s Veterinary Organization revised its avian influenza control and prevention policy to include mass vaccination campaigns using H5 inactivated vaccines in the AI control programs.

This research establishes the serological baseline of antibodies produced by H5 vaccines. We also examined the durability and stability of the mean titer of protective antibodies induced by vaccination (4.4 <). Our antigen was prepared from the H5N8 virus isolated in Iran (A/Chicken/Iran/162/2016(H5N8)) clade 2.3.4.4b. Molecular detection via quantitative reverse transcription polymerase chain reaction (qRRT-PCR) was used to ensure the flock was free from any possible H5 challenge. Additionally, we evaluated and measured the neutralizing ability of antibody-containing sera caused by different vaccines.

METHODS

Selection of pullet and layer flocks

We selected ten layer-type pullet rearing farms from different geographical areas of Tehran province, totaling 60 houses with a population of about 1,500,000 commercial layers with a permit, sanitary, and structural conditions needed by this research. At least 30 chickens from each house were identified as sentinel and non-vaccinated H5 (negative control) groups. Approximately 800,000 out of 1,500,000 pullets were transferred to 8 different layer farms across 26 houses (the population of each pullet farm to a layer farm separately), while maintaining the non-vaccinated control groups. These groups were subjected to continuous monitoring and research studies during the production period.

Vaccines and vaccination

Vaccine A: Inactivated hemagglutinin subunit vaccine (H5N1) by Boehringer Ingelheim (Germany), derived from a baculovirus with inactivated LaSota strain of Newcastle virus. Seed: Influenza A virus (A/duck/China/E319-2/03(H5N1)) clade 2.3.2.

Vaccine B: Inactivated reverse genetic recombinant subunit H5N3 vaccine by Zoetis Inc. (USA). Seed: Influenza A virus (A/chicken/Vietnam/C58/04(H5N1)) clade 1.

Vaccine C: Inactivated Re6+Re8 vaccine with a rearranged genome using reverse genetics by Harbin Veterinary Research Institute (China). Seed: Influenza A virus Re6 (A/duck/Guangdong/23/2004(H5N1)) clade 2.3.2.1b and Re8 (A/chicken/Guizhou/4/13(H5N1)) clade 2.3.4.4b.

We tested each vaccine on at least two separate farms. After separating the control chickens in each house, technicians injected the remaining chickens subcutaneously with 0.5 cc of the vaccine in the back of the neck using an automatic syringe. Vaccination occurred in two rounds: first at 30–45 days old and the second four weeks later.

Sampling

Blood sampling

To evaluate the serological performance of vaccines, each farm was sampled 7–8 times. Each sampling included 22 blood samples from sentinel groups, 22 from vaccinated birds of each house, and 22 to 90 blood samples of each layer house (depending on the population of birds in the house during the laying period). We collected a total of 11,308 serum samples over seven periods: 1,320 before vaccination, 2,640 before the second vaccine, 2,002 four weeks after the second vaccine, 1,276 after transfer to the laying unit (20–24 weeks), 1,276 at production period of 29–33 weeks, 1,276 at production period of 37–43 weeks, 1,276 at production period of 50–53 weeks, and 242 at 63 weeks). These samples were stored for hemagglutination inhibition (HI) testing.

Swab sampling

To molecularly trace HPAI viruses in the farms and verify flock cleanness before vaccination, as well as to store samples for polymerase chain reaction (PCR) testing in case of unexpected changes in serological test results, 6,420 tracheal and cloacal swab samples were collected simultaneously or up to 2 weeks before each serum sampling.

Molecular diagnosis test (PCR)

Before the initial vaccination round, samples were collected from both the trachea and cloaca, along with 8 out of 27 layer houses experiencing a production decrease of 10%–12%. RNA extraction was conducted using the Sina Pure RNA Extraction Kit per its brochure. Molecular detection of influenza virus RNA was then carried out using the H5-specific qRRT-PCR method [14]. Additionally, H9 molecular detection was conducted on pre-H5 vaccination swabs using the H5 vaccine.

Hemagglutination inhibition (HI β) test

The HI β test was performed according to references using a non-homologous vaccine antigen prepared from H5N8 (A/Chicken/Iran/162/2016(H5N8)) clade 2.3.4.4b isolated in Iran [16]. Additionally, the HI test was performed with H9 commercial antigen on the serum of blood samples taken before starting H5 vaccination.

Determining the neutralizing potency of serum antibodies

The sera from vaccine groups A, B, and C (whose titers were in the range of their respective baselines obtained in this study), collected four weeks after the second round of vaccination from the farms, and the control groups were collected separately. After re-determining the titer (HI β test) of pooled sera for each group, HI α (in vitro) and SN (in vivo) were used to determine the neutralizing potency of antibodies produced after each vaccine.

Serum titration test by HI α method

In the α method of the HI test, which was performed for pooled sera from each vaccine group and the control group, unlike the HI β method, a series of antigen dilutions is prepared, and a fixed amount of serum is added to the wells. The rest of the steps are similar.

Serum neutralization (SN) test

For each serum group (control, vaccine A, B, C), six sterile tubes were prepared. Each tube received 0.5 cc of serum, previously kept in a bain-marie at 56°C for 45 min and centrifuged at 2,000 rpm for 5 min. Then, 0.5 cc of the serum was added to each of the six tubes containing serial logarithmic dilutions (−3 to −8) of the H5 virus. The virus-serum mix was incubated at 37°C for 45 min. After every six dilutions, 0.2 cc of each serum-virus mix was injected into four 9–11-day-old embryonated eggs from healthy breeder farms (no H5 vaccination or disease history). The eggs were prepared, checked for viability, marked, disinfected, and injected through the allantoic sac. Allantoic fluid (at least 0.5 cc per egg) was collected and stored for the HA test after 48–72 h of incubation at 37°C.

At the same time, to determine the 50% lethal dose (LD50) of the H5 virus used in the SN test, each of the −3 to −8 dilutions (prepared from the virus without mixing with serum) was injected into the allantoic sacs of four embryonated eggs. After 48–72 h of incubation at 37°C and overnight refrigeration at 4°C in a sterile manner, samples were collected from each egg, stored in microtubes, and frozen for the HA test. After thawing and centrifugation of all the collected and stored allantoic fluids, the HA test was conducted on all samples [14].

RESULTS

PCR test

To ensure the chicken population was clean pre-vaccination, molecular testing (qRRT-PCR) was conducted on tracheal and cloacal swab samples, yielding negative results for the HPAI H5 subtype. Subsequent cloacal samples, collected post-vaccination or during production, showed no increase in serum titers or any positive titers in the control group. They also showed the absence of disease symptoms. Swab samples from 8 out of 27 houses, reporting a 10%–12% reduction in production, underwent molecular tracking via qRRT-PCR to confirm the absence of viral challenge. The swab sample extract was then injected into 9–10-day-old embryonated eggs and underwent passage up to three times. Direct PCR results of both the swabs and embryonated eggs yielded negative results for HPAI. Additionally, all pre-vaccination swabs tested negative for H9 subtype by PCR.

HI β test

During the HI β test, the geometric mean titer log2 > 4.4–5 was set as a criterion for serum monitoring post-vaccination, serving as an effective target titer for reducing virus shedding [16,17].

Vaccine A was studied in farms 1–3, vaccine B in farms 4–7 and vaccine C in farms 8–10. As shown in Table 1, the geometric mean of antibody titers is based on log2. The results of sera obtained from injections of vaccines A, B or C in farms 1–10 (ages 4 to 63 weeks) are also shown in Table 1.

Table 1. The geometric mean titer of HI against the antibody of vaccines A, B and C of blood serum samples of 10 different farms.

Farm No. (houses)	Vaccine type	Time of sampling
		Before of Vaccination (age: 4–8 wk)		4 wk post first vaccinatio (age: 8–12 wk)		4 wk post second vaccination (age: 12–16 wk)		At 20–24 wk		At 29–33 wk		At 37–42 wk		At 50–53 wk		At 63 wk
		Number of samples	Mean titers	Number of samples	Mean titers (min–max)	Number of samples	Mean titers (min–max)	Number of samples	Mean titers (min–max)	Number of samples	Mean titers (min–max)	Number of samples	Mean titers (min–max)	Number of samples	Mean titers (min–max)	Number of samples	Mean titers (min–max)
1(3)b	A	66 (3*22)	Negative	66 (3*22)	2.58 (2.1–3.15)	66 (3*22)	5.2 (4.9–5.6)	66 (3*22)	4.66 (4.3–4.7)	66 (3*22)	4.32 (3.93–4.5)	66 (3*22)	3.81 (3.65–4)	66 (3*22)	0.75 (0.6–0.85)
2(8)b	A	176 C6:C19 (8*22)	Negative	176 (8*22)	2.99 (2.7–3.25)	176 (8*22)	4.83 (4.2–5.3)	176 (8*22)	4.99 (4.7–5.25)	176 (8*22)	3.28 (3.1–3.75)	176 (8*22)	2.07 (1.8–2.6)	176 (8*22)	0.35 (0–0.5)
3(8)b	A	176 (8*22)	Negative	176 (8*22)	2.85 (2.5–3.2)	176 (8*22)	4.9 (4.5–5.25)	a	a	a	a	a	a	a	a
1, 2, 3(19)	A	418 (19*22)	Negative	418 (19*22)	2.8 (2.58–2.99)	418 (19*22)	4.97 (4.83–5.2)	242 (11*22)	4.825 (4.66–4.99)	242 (11*22)	3.8 (3.28–4.32)	242 (11*22)	2.94 (2.07–3.81)	242 (11*22)	0.55 (0.75–0.35)
4(2)b	B	44 (2*22)	Negative	44 (2*22)	4.4 (4.05–4.8)	44 (2*22)	5.92 (5.3–6.55)	44 (2*22)	5.1 (5.1–5.1)	44 (2*22)	4.5 (4.3–4.7)	44 (2*22)	4.1 (3.7–4.5)	44 (2*22)	2.95 (2.3–3.6)
5(4)b	B	88 (4*22)	Negative	88 (4*22)	5.33 (4.55–5.9)	88 (4*22)	6.44 (6–7.35)	88 (4*22)	5.65 (5.3–6.1)	88 (4*22)	5.375 (4.95–5.35)	88 (4*22)	4.05 (3.85–4.3)	88 (4*22)	2.4 (2.15–2.55)
6(3)b	B	66 (3*22)	Negative	66 (3*22)	5.42 (5.15–5.8)	66 (3*22)	6.4 (6–6.9)	66 (3*22)	5.8 (5.25–6.2)	66 (3*22)	5.4 (5.15–5.65)	66 (3*22)	4.07 (3.9–4.25)	66 (3*22)	2.5 (2.4–2.6)
7(4)b	B	88 (4*22)	Negative	88 (4*22)	5.19 (4.9–5.65)	88 (4*22)	6.08 (5.9–6.15)	88 (4*22)	6 (5.9–6.05)	88 (4*22)	5.7 (5.55–6)	88 (4*22)	5.19 (4.9–5.15)	88 (4*22)	3.35 (2.9–3.75)
4, 5, 6, 7(13)	B	286 (13*22)	Negative	286 (13*22)	5.085 (4.4–5.42)	286 (13*22)	6.2 (5.92–6.44)	286 (13*22)	5.64 (5.1–6)	286 (13*22)	5.09 (4.5–5.5)	286 (13*22)	4.51 (4.05–5.19)	286 (13*22)	2.8 (2.4–3.35)
8(10)b	C	220 (10*22)	Negative	220 (10*22)	5.2 (4.3–5.6)	88 (1*88)	8.75	88 (1*88)	8.8	88 (1*88)	8.5	88 (1*88)	8	88 (1*88)	7.49	88 (1*88)	5.78
9(10)b	C	220 (10*22)	Negative	220 (10*22)	4.7 (4.35–5.05)	88 (2*44)	9.12 (8.94–9.3)	88 (2*44)	9.1 (8.5–9.3)	88 (2*44)	8.86 (8.94–8.78)	88 (2*44)	8.44 (8.6–8.3)	88 (2*44)	7.1 (7.3–6.9)	88 (2*44)	5.85 (6.1–5.6)
10(8)b	C	176 (8*22)	Negative	176 (8*22)	5.24 (5–5.4)	176 (8*22)	8.4 (8.1–8.75)	a	a	a	a	a	a	a	a	a	a
8, 9, 10(28)	C	616 (28*22)	Negative	616 (28*22)	5.04 (4.7–5.24)	352	8.76 (8.4–9.12)	176	8.95 (8.8–9.1)	176	8.68 (8.5–8.86)	176	8.22 (8–8.44)	176	7.3 (7.1–7.49)	176	5.8 (5.75–5.85)

HI titer was not detected in any sera obtained from the control groups at any sampling times and in any of the samples (total samples = 5,588).

HI, hemagglutination inhibition.

^aAfter 12 weeks, due to the handing over and transfer of the pullets to the out-of-province unit and the impossibility of direct supervision of this farm, it was excluded from further study during the production period. It was excluded from further study during the production period.

^bSerology of chickens vaccinated with vaccine A, B or C (HI titer monitoring against the antigen prepared from H5N8 virus isolated in Iran) in farms 1–10 by houses. Titers are based on Log2 geometric mean titers ± standard error. Statistical analysis with two-way analysis of variance does not show a significant difference between the results of the houses in each farm:

Farm 1 houses(1–3): p = 0.467; Farm 2 houses(1–8): p = 0.699; Farm 3 houses(1–8): p = 0.074; Farm 4 houses(1–2): p = 0.778; Farm 5 houses(1–4): p = 0.099; Farm 6 houses(1–3): p = 0.099; Farm 7 houses(1–4): p = 0.862; Farm 8 houses(1–10): p = 0.50; Farm 9 houses(1–10): p = 0.45; Farm 10 houses(1–8): p = 0.52.

The highest average of mean titers for vaccine A (farms 1–3) was 4.97, corresponding to the samples taken four weeks after the second round of the vaccination, stable (log2 > 4.4–5) until the age of 25–30 weeks (Fig. 1A).

Fig. 1. Comparative chart of all farms using vaccine A, B, and C and comparative chart of serums using these three different vaccines.

(A) Comparative chart of all farms using vaccine A. This part presents a comparative chart of the geometric mean titers obtained from serum samples of farms 1, 2, and 3, where vaccine A was used. The x-axis represents the age range at sampling times, which varies across different farms. Titers are based on Log2 GMT ± SE. Statistical analysis with two-way ANOVA does not show a significant difference between the results of the houses (p = 0.882). (B) Comparative chart of all farms using vaccine B. This part presents a comparative chart of the geometric mean titers obtained from the serum samples of farms 4, 5, 6, and 7, where vaccine B was used. The x-axis represents the age range at sampling times, which varies across different farms. Statistical analysis with two-way ANOVA does not show a significant difference between the results of the houses (p = 0.101). (C) Comparative chart of all farms using vaccine C. This part presents a comparative chart of the geometric mean titers obtained from the serum samples of farms 8, 9, and 10, where vaccine C was used. The x-axis represents the age range at sampling times, which varies across different farms. Statistical analysis with two-way ANOVA does not show a significant difference between the results of the houses (p = 0.931). (D) Comparative chart of serums using three different vaccines. This part compiles the graphs from Fig. 1A-C into a comparative chart showing the geometric mean of the serum titers obtained from the separate use of vaccines A, B, and C. The x-axis represents the age range at sampling times, which varies across different farms. Statistical analysis with two-way ANOVA shows a significant difference between the results of the houses (p = 0.02).

GMT, geometric mean titers; SE, standard error; ANOVA, analysis of variance.

The highest average of mean titers for vaccine B (farms 4–7) was 6.2, corresponding to the samples taken four weeks after the second round of the vaccination, stable (log2 > 4.4–5) until the age of 35–45 weeks (Fig. 1B).

The highest average of mean titers for vaccine C (farms 8–10) was 8.57, corresponding to the samples taken four weeks after the second round of vaccination, stable (log2 > 5–4.4) until the age of 63 weeks (Fig. 1C).

In all sera obtained from the control groups (sentinel) and in all the sampling sessions, no H5 HI titers were detected.

According to the HI β test results performed with the H5N8 antigen of the Iranian isolate in this study, the baselines are the average antibody titer resulting from two rounds of vaccination (4 weeks after the second round) with A, B, and C vaccines based on log2 in the range of 4.83–5.2, 5.9–6.43, and 8.4–9.12, respectively. The stability of the geometric mean titer was log2 > 4.4–5 until 25–30 weeks, 35–45 weeks, and above 63 weeks, respectively (Table 1, Fig. 1D).

The HI test results using commercial H9 antigen on pre-H5 vaccination serum samples from all flocks showed titers of 3–4 (results not shown). All flocks had received a single dose of the H9 vaccine before sampling.

Tests to determine the neutralizing potency of serum antibodies

Pooled serum samples from vaccine groups A (serum 1), B (serum 2), C (serum 3), and the control group (serum 0) were utilized in HI α and SN tests to determine neutralizing potency. Their respective titers in HI β were 5, 6, 8, and 0.

Serum titration test by HI α method

The HI α test used a fixed amount of serum from each of the pooled serum groups against serial logarithmic base 2 dilutions with a non-homologous H5N8 antigen (A/Chicken/Iran/162/2016(H5N8), clade 2.3.4.4b) to determine neutralizing potency. It was observed that serum 3, despite the higher titer of 8 in the HI β test (compared to both other sera), showed similar results to serum 2, reducing antigen potency by 5 logarithms compared to the serum of the sentinel group, while serum 1 reduced only 3 logarithms of the antigen potency.

SN test

Serial dilutions of the virus, ranging from −1 to −6, were prepared, and a fixed amount of serum was added to each of the sera 1, 2, 3, and 0. Each mixture was then injected into the allantoic fluid of four 9–10-day-old embryonated eggs and left to incubate at 37°C for 48–72 h. Subsequently, all allantoic fluids were collected for HA testing. The results, displayed in Table 2, were utilized to calculate the 50% protective dose and the neutralization index (NI) of each serum (sera 1, 2, 3) by the Reed & Muench method (Table 3). Simultaneously, the LD50 of the virus was determined by injecting dilutions ranging from −3 to −8 into the allantoic fluid of 9–10-day-old embryonated eggs and performing HA testing (Table 4). The LD50 was found to be 10^7.7. An overview of the test results on the serums is provided comparatively in Table 5.

Table 2. Allantoic fluid HA results from SN test for each dilution (fixed serum + virus with successive dilutions) injected into four 9–10 days old embryonated eggs.

LD50 of the virus		Serum 1		Serum 2		Serum 3
Dilution	Result	Dilution	Result	Dilution	Result	Dilution	Result
−3	+4	−1	+4	−1	+3	−1	+2
−4	+3	−2	+3	−2	+2	−2	+1
−5	+4	−3	+1	−3	+1	−3	0
−6	+3	−4	0	−4	0	−4	0
−7	+2	−5	0	−5	0	−5	0
−8	+1	−6	0	−6	0	−6	0

HA results for serum zero (control) allantoic samples are not listed in the table.

HA, hemagglutinin; SN, serum neutralization; LD50, 50% lethal dose.

Table 3. PD50 of sera.

Serum No.	Dilution	Positive result	Negative result	Cumulative number of positive results (+/+)	Cumulative number of negative results (+/−)	Relative positive results to total results	Positive result percentage
1a	−1	4	0	8	0	8/8	100%
	−2	3	1	4	1	4/5	80%
	−3	1	3	1	4	1/5	20%
	−4	0	4	0	8	0/8	0%
2b	−1	3	1	6	1	6/7	86%
	−2	2	2	3	3	3/6	50%
	−3	1	3	1	6	1/7	14%
	−4	0	4	0	10	0/10	0%
3c	−1	2	2	3	2	3/5	60%
	−2	1	3	1	5	1/5	17%
	−3	0	4	0	9	0/9	0%

PD50, 50% protective dose.

^aSerum 1:

$$Proportionate\hspace{0.33em}Distance\frac{upper50\text{\%}-50\text{\%}}{upper50\text{\%}-lower50\text{\%}}=\frac{80-50}{80-20}=\frac{30}{60}=0.5$$

T = 10^2.5 × 10 = 10^3.5

Neutralization Index (NI) = Logarithm of Virus Titer − Logarithm of Mix Titer of Serum and Virus

Serum1 = log(10^7.7) − log(10^3.5) = log(10^4.2) = 4.2

^bSerum 2:

$$Proportionate\hspace{0.33em}distance\frac{upper50\text{\%}-50\text{\%}}{upper50\text{\%}-lower50\text{\%}}$$

T = 10² × 10 = 10³

Neutralization Index (NI) = Logarithm of Virus Titer − Logarithm of Mix Titer of Serum and Virus

Serum2 = log(10^7.7) − log(10³) = log(10^4.7) = 4.7

^cSerum 3:

$$Proportionate\hspace{0.33em}Distance\frac{upper50\text{\%}-50\text{\%}}{upper50\text{\%}-lower50\text{\%}}=\frac{60-50}{60-17}=\frac{10}{43}=0.2$$

T = 10^2.2 × 10 = 10^3.2

Neutralization Index (NI) = Logarithm of Virus Titer − Logarithm of Mix Titer of Serum and Virus

Serum3 = log(10^7.7) − log(10^3.2) = log(10^4.5) = 4.5

Table 4. Calculating LD50 of the virus.

Dilution	Positive result	Negative result	Cumulative number of positive results (+/+)	Cumulative number of negative results (+/−)	Relative positive results to total results	Positive result percentage
−3	4	0	17	0	17/17	100%
−4	3	1	13	1	13/14	92.85%
−5	4	0	10	1	10/11	90.09%
−6	3	1	6	2	6/8	75%
−7	2	2	3	4	3/7	42.85%
−8	1	3	1	7	1/8	12.50%

LD50, 50% lethal dose.

$$Proportionate\hspace{0.33em}Distance\frac{upper50\text{\%}-50\text{\%}}{upper50\text{\%}-lower50\text{\%}}=\frac{75-50}{75-42.85}=\frac{25}{32.15}=0.$$

T = 10^6.7 × 10 = 10^7.7

Table 5. Overall results of all tests on serums.

Serum type	Average of HI β titer post first vaccination	Average of HI β titer post second vaccination	Age of antibody titer stability (4.4 <)	HI α	NI
Vaccine A	2.58–2.99	4.83–5.2	25–30 weeks	5	104.2
Vaccine B	4.42–5.42	5.9–6.43	35–45 weeks	3	104.7
Vaccine C	5.2 Mehdi Vasfi Marandi –5.24	8.4–9.12	More than 60 weeks	3	104.5
A, B, and C control	0	0	-	8	-

HI, hemagglutination inhibition; NI, neutralization index.

DISCUSSION

The influenza virus has long posed a persistent threat to public health and the poultry industry. With its genetic mutations (shift and drift), the virus has capitalized on the global proliferation of poultry farms and other types of birds, especially aquatic species. This increased circulation, even across species, is facilitated by reduced geographical distances between habitats and their breeding places. Consequently, the virus moves more freely between wild and domestic bird populations, leading to virus passaging and genetic rearrangements in different hosts [18]. Today, we confront genetic changes and the emergence of new behaviors in this highly contagious virus more frequently and rapidly than ever before. Recent years have seen a surge in epidemics and the resurgence of HPAI in the northern hemisphere, with reports now also emerging from the southern hemisphere, notably South America [18,19].

Over the past two decades, some countries have adopted ring vaccination as a targeted short-term strategy against HPAI outbreaks, aiming to control the disease and minimize virus shedding while eradicating affected populations at the disease center. However, mass vaccination has been adopted as a strategic approach in Southeast Asia (also China) and the Middle East (Egypt, Iran) due to the endemicity of the disease and its rapid spread, respectively [9]. Globally, 95.5% of vaccine usage is inactivated oil-emulsion, with only 4.5% being recombinant [20].

The serum level of anti-hemagglutinin antibodies measured by the HI test is used to evaluate the vaccine’s effectiveness. Evidence strongly shows an association between HI levels and protection after the administration of inactivated vaccines [21]. Additionally, virus shedding and transmission reduce with HI titers above four [16], and no virus shedding was observed with HI titers above 5 [22]. Despite this, the minimum antibody titer with the HI test may not apply to recombinant vector vaccines [17,23,24].

Kwon et al. [25], in a study comparing the effectiveness of two recombinant rHVT-H5 vaccines and inactivated RE-6 vaccines, showed that while there was no significant difference in HI titers with the homologous antigen post-vaccination. the birds vaccinated with the RE-6 vaccine had significantly higher HI antibody titers with the challenge virus antigen. Additionally, the cross-reactivity of antibodies caused by the rHVT-H5 vaccine against circulating viruses in Bangladesh was lower.

Another study by Astemirov et al. [26] on an inactivated recombinant vaccine derived from a dual inactivated recombinant AI+ND baculovirus-derived vaccine and an inactivated whole H5N1 virus vaccine showed that the HI test results 30 days after a single dose of vaccination for the inactivated vaccine (H5N1) showed little difference between homologous (titer six on the log base 2) and recombinant (titer eight on the log base 2) antigens. However, for the inactivated recombinant vaccine derived from baculovirus, the results differed significantly between the two antigens (p = 0.003), showing a strong humoral immune response with recombinant antigen (titer nine on the log base 2) in contrast to a mild reaction with non-recombinant antigen H5N1 (titer three on the log base 2). Given that the homologous antigen did not significantly differ in the humoral immune response of both vaccines, the inactivated H5N1 whole virus vaccine was able to produce a higher level of HI antibodies (titer greater than eight on the log base 2 scale) and increase the serum level to 88%, 30 days after a single dose of vaccination. In contrast, birds that received the recombinant baculovirus vaccine, showed only a 13% increase in serum levels, with the highest HI titer being five on a log base 2 scale [26].

In our study, the geometric mean antibody titer against the antigen prepared from the H5N8 virus isolated from Iran (with a homology of 91.27% to 92.66% with the vaccine seeds) after a single vaccination for the sera obtained from the inactivated hemagglutinin subunit vaccine derived from baculovirus with the inactivated LaSota strain of Newcastle virus was 2.87. In contrast, the sera antibodies from the inactivated recombinant reverse genetic vaccine subtype H5N3 had a titer of 5.2, and the inactivated H5 recombinant reverse genetics Re6+Re8 had a titer of 5.03. After two vaccinations, the results were 4.9, 6.25, and 8.57, respectively.

The studies of Yuk et al. [27] in Vietnam and Abdelwhab and Hafez [28] in South Africa indicate that no serum titer was detected in HI tests using the standard method after administering recombinant vaccines. However, when the recombinant (homologous) antigen was used in the serum of chickens vaccinated with the recombinant H5 derived from baculovirus, the average titer was 8.6 [27,28]. Previously, Qiao et al. [29,30] demonstrated that an inconsistent or low antibody response after vaccination of chickens with a recombinant avian influenza vaccine may be related to the antigen used in the HI test. Of course, the effectiveness of this recombinant vaccine, among other factors (such as the type of antigen used in HI), also depends on the carrier vector [31,32]. For example, recombinant vaccines containing the H5 antigen expressed in baculovirus may show differences in glycosylation between insect and bird cells, potentially affecting the antibody titer in vaccinated birds.

The results of HI by Kapczynski et al. [15] in the US on serum samples obtained from the blood of chickens vaccinated with inactivated or recombinant vaccines (each group having a different form of their HA antigen) using two vaccine antigens and a challenge antigen (prepared from A/gyrfalcon/Washington/40188-6/2014 H5N8 clade 2.3.4.4 virus) show a significant difference in titration with two different antigens.

It is generally accepted that the presence of hemagglutination inhibitory antibodies has a high predictive value for protection against mortality when challenged with the virus [33,34,35]. Nevertheless, the absence of these antibodies does not necessarily indicate a lack of mortality prevention and has a low predictive value [35].

The HI test primarily detects antibodies bound to the receptor site, potentially overlooking virus-neutralizing antibodies in the hemagglutinin stalk. These antibodies interfere with pH-induced conformational changes linked to membrane fusion or neuraminidase activity. Despite the absence of HI antibody titers, vaccinated chickens may still be protected due to antibodies targeting the hemagglutinin stem. Recent research has identified protective epitopes on the stem and other hemagglutinin regions in mammals, where neutralizing antibodies operate independently of HI activity [36,37].

Considering the above information and the ongoing validity of the HI test in assessing avian humoral immune response to inactivated vaccines by quantifying antibodies, we conducted this study to establish a baseline for serological comparison among three imported inactivated vaccines utilizing varying production technologies. Also, determining the stability time of the protective antibody titer resulting from vaccination can be very useful to revise disease prevention and control programs. We employed a fixed domestic HPAI (H5N8) antigen isolated from Iran (A/Chicken/Iran/162/2016(H5N8)) clade 2.3.4.4b in our study. This ensures that the HI test titer closely reflects the protective capacity of vaccinated chickens against probable challenge viruses and evaluates the neutralizing potency of each vaccine’s pooled sera (after determining the updated titer) using both in vitro (HI α) and in vivo (SN) methods. The HA gene homology between the antigen we used and each seed in vaccines A, B, and C (Re6+Re8) is ‘92.66%,’ ‘91.27%,’ and ‘91.86% and 91.96%,’ respectively. Assessing the neutralization coefficient of serum-neutralizing antibodies via the SN test can indicate the vaccine’s efficacy against the dominant pathogenic virus in circulation. Utilizing this test in embryonated eggs demonstrates a stronger correlation with experimental or natural challenge conditions in vaccinated chickens. It was concluded that after two times vaccination of the H5N3 baculovirus, monovalent H5N3 vaccine, and recombination vaccine Re6+Re8 in laying hens, the serology baseline was different in Iran. The serum antibody titer of the Re6+Re8 vaccine in the HI β assay with the H5N8, isolate from Iran (A/Chicken/Iran/162/2016(H5N8)) clade 2.3.4.4b showed a higher and more stable numerical titer compared to the H5N3 vaccine and baculovirus vaccine. However, NI for H5N3 vaccine was 0.2 higher than Re6+Re8 and 0.5 higher than baculovirus vaccines.