Improvement Of Inactivated Equine Herpes Virus-1 Vaccine Using Carbomer

An inactivated Equine herpes virus-1 vaccine was successfully improved using carbomer as adjuvant inducing high and long immunity in vaccinated mares in comparison with the convention one adjuvanted with Al-hydra gel and saponin. Such purpose was established by using 0.5% carbomer as adjuvant to the inactivated EHV-1. The applied quality control tests carried out on such vaccine revealed that it is free from foreign contaminants, safe in pregnant mares and mice and potent induced high levels of specific EHV-1 antibodies in vaccinated Guinea pigs and mares as measure by ELISA and SNT. This immunity was sufficient to protect vaccinated horses up to 28 weeks (7months) post-vaccination. ـ ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ


INTRODUCTION
Equine herpesvirus type 1 (EAV-1) causes abroad range of manifestations in horses, including a central nervous system disease involving the spinal cord and brain (myeloid cephalopathy), respiratory disease, abortions and perinatal death (Meaquita et al., 2017). Abortion may result from exogenous or endogenous infection, i.e., reactivation of latent virus (Allen et al., 1998).
In recent years, increased incidence of Equine herpesvirus myeloid Cephalopathy (EHM) has been observed by infection with the same virulent strains of EHHV-1 leading to inflammation of the blood vessels supply the brain and spinal cord (Henninger et al.,  2007). Control of EHV-1 infection is so tricky that the symptoms may be subclinical and may be reactivated after many months or years after primary infection under stress factors (Browning et al., 1988). Vaccination is crucial to control EHV-1 infection using either live-attenuated or inactivated vaccines with the inactive one's preferable use (Mayer et al., 1978).
In inactivated vaccines, an ideal adjuvant should be safe, stable, bio gradable and ensure vaccine potency's reproducibility during manufacture (Cater and Reed, 2010). Carbomer has low reactivity, no virucidal nature and efficacy in one shot vaccination schedules; a lightly cross-linked polymer of acrylic has become widely used as an adjuvant in the veterinary field (Diamantstein et al., 1971) enhance the strength and duration of antibody responses stimulated by inactivated equine influenza vaccines as compared with vaccines of equivalent antigenic content (Wood et al., 1983). Production of GHPV antigen inactivated adjuvanted vaccine (Jac Queline et al., 2010) vaccine induces a robust serological response in growing gosling and breeder. Carbomer promotes an early onset of cellular immunity by facilitating the cell differentiation towards effector phenotypes and efficiently inducing native to memory transition (Mair et al., 2015). Using carbomer in animal models results in adjuvant system activity, including strong proinflammatory type-1 T cell (TH1) polarization (Schwabe et al., 1977 However, the cellular immune response is also essential in pregnant mares, positive correlation between the frequency of EHV-1 CTL (cytotoxic Tlymphocyte) and the protection against the disease (Kydd et al., 2003). This study was designed to prepare and evaluate the safety and potency of inactivated EHV-1 adjuvanted with carbomer and a mixture of saponin as vaccine adjuvants.

Equine herpes virus-1 (EHV-1)
Locally isolated EHV-1 isolated by Hassanein et al. (2002) and adapted on Vero cell culture by Safaa (2007) identified by reference freeze-dried rabbit anti-EHV-1 (kindly supplied by Dr Jennet Wellington, Research Follow Department of Biological Science, Macquarie Univ., NSW Australia) was supplied by the Department of Equine Vaccine Research (DEVR); Veterinary Serum and Vaccine Research Institute (VSVRI) and used for an inactivated vaccine.

EHV-1 antiserum
Locally prepared EHV-1 antiserum (Safaa et al., 2005) was supplied by DEVR and used as a positive control in the serological tests.

Experimental animals 3.1. Mares
Eleven health adult mares were used in the present study where 3 of them were used to test the safety, and six mares were used to test the potency of the prepared vaccine while two mares were kept without vaccinates as a test control. All mares were housed under hygienic measures receiving balanced ration and adequate water following animal ethics.

Mice
Three groups of pregnant Swiss Albino mice of 4-6 weeks old (4mice/ group) were used for safety testing of the prepared inactivated EHV-1 vaccine

Guinea pigs
Twelve seronegative Guinea pigs of about 300-400gm body weight were divided into three groups were the first two groups were used to assess the potency of the carbomer and Al-hydra gel with saponin adjuvanted vaccines respectively, keeping the third group without inoculation as test control and the second group was used 4. Cell culture African green monkey kidney cell line (Vero) was maintained and propagated using Minimum Essential medium with Eagle's salts and used to prepare EHV-1 suspension, virus titration, and the residual viral testing inactivated virus suspension and serum neutralization test.

Embryonated chicken eggs
Specific pathogen-free Embryonated chicken eggs (SPF-ECE) of 11-13 days old were obtained from SPF eggs farm Koum Osheim, Fayoum Governorate, Egypt and used for assurance of complete virus inactivation

Saponin
Saponin was obtained from Ubichem. PLC as powder prepared as a solution of 2mg/ml of purified saponin in distilled water with PH 7.5 and sterilized by autoclaving at 108 o C for 15 minutes according to Hamdy (2016)

Preparation of carbomer solution
Carbomer was supplied from Lubrizol Co. as a fluffy white powder. It was dissolved in hot water to prepare 0.5% aqueous stock solutions, sterilizes by autoclaving at 121 o C under the pressure of 1.5lb/ inch for 20 min, then stored at 4 o C until use (United States pharma Cofield Convention, 1990).

Preparation of EHV-1 suspension
The local EHV-1 was propagated in three successive passages in Vero cells. The virus suspension was collected and clarified by centrifugation at 3000 rpm for 15 minutes, then titrated where the virus titer was calculated according to Reed and Muench (1938). It was recommended that the virus titer should not be less than 7log10 TCID 50 / ml for preparation of inactivated EHV-1 vaccine (OIE, 1990).

Detection of residual infective virus
This step was carried out through: 10.1. Inoculation of the chorioallantoic membrane of ECE incubated at 37 o C for five days with daily examination for detection of pock lesions, which should not be detected as recommended by Safaa and Hussein (2012). 10.2. Inoculation of Vero cells to ensure the absence of EHV-1 CPE through three blind passages according to OIE (2017)

11.Vaccine preparation
Two batches of inactivated EHV-1 vaccine were prepared where one of them was 20% of Al-hydra gel and the second one was mixed in equal volumes carbomer according to Naglaa et al. (2020).

Quality control testing of the prepared vaccine 12.1.Sterility test
Random samples of the prepared inactivated EHV-1 vaccine with carbomer were cultured on different media to ensure the vaccine freedom of foreign contaminants (aerobic and anaerobic bacteria, fungi and mycoplasma) according to the recommendations of OIE (2017) 12.2. Saftey test 12.

In mice
This test was performed in 2 groups of pregnant mice (10 mice/group) where the first group was inoculated I/N with 45µl of the inactivated virus/ mouse while the 2 nd group was inoculated S/C with 0.3ml of the prepared vaccine/ mouse according to Kirisawa et al. (1995) and kept under a hygienic condition and daily observation for two weeks

In mares
Each of three mares was inoculated I/M with a dose of 2ml of one of the prepared vaccines/mares receiving a second dose one month later. These mares were kept under hygienic measures for two weeks.

Potency test 12.2.3.1. In Guinea pigs
This test included three groups of Guinea pigs of about 300-400 gm body weight (3 animals/group). The first group was inoculated S/C with a 0.2ml/ animal of the inactivated EHV-1 vaccine with rehydrated gel and saponin. The second group was inoculated in the same manner with the same dose of the prepared vaccine with carbomer. Each group received a second dose after one week. The third group was kept without vaccination as test control and serum samples were obtained from all groups on the 3 rd and 5 th -week post-vaccination.

In mares
Eight healthy mares with low EHV-1 antibody titer (≤4 NI) were divided into three groups as follow: Group-A of 3 mares were vaccinated I/M with a dose of 2ml of Al-hydra gel and saponin adjuvanted vaccine/ mare Group-B of 3 mares were vaccinated I/M with a dose of 2ml of carbomer adjuvanted vaccine/ mare Group-C of 2 mares was kept without vaccination as test control Each of the vaccinated groups received a booster of the corresponding vaccine four weeks post the first vaccination.
Serum samples were obtained from all mares on 2-week interval up to 28 weeks post-vaccination to follow up the induced levels of EHV-1 antibodies in vaccinated mares using serum neutralization test and ELISA

Serum neutralization test (SNT)
SNT was carried out on vaccinated mare sera and the antibody titer was expressed as serum neutralization index (NI) according to Shanker et al. (1989) and Hamdy (2016).

RESULTS
The applied quality control testes on the prepared vaccine revealed that it is free from foreign contaminants (aerobic and anaerobic bacteria; fungi and mycoplasma); safe inducing no abnormal local or systemic post inoculation reactions in Guinea pigs and mares and potent providing vaccinated animals with good levels of specific EHV-1 antibodies as shown in table (1). Application of indirect ELISA on serum samples of vaccinated Guinea pigs showed that these animals exhibited antibody titers 400 and 490 by the 3 rd week post vaccination in those vaccinated with the alhydra gel vaccine and those vaccinated with the carbomer vaccine reached to titers of 940 and 1019 by the 5 th week post vaccination respectively (table 2). Regarding vaccination of mares tables (3 and 4) showed that the inactivated EHV-1 vaccine adjuvanted with carbomer induced higher ELISA titer (1480) by the 6 th week post vaccination while the gel vaccine showed ELISA titer (1360) on the same period with SNI 2.0 and 1.9 by the 2 vaccines respectively. Peak titers (1690 and 1595 by ELISA and 3.5 and 3.2 SNI respectively by the 10 th week recording their lowest levels (803 and 770 by ELISA and 1.8 and 1.3 SNI respectively by the 28 th week.

DISCUSSION
The present study was performed for the preparation and evaluation of inactivated EHV-1 vaccine adjuvanted with carbomer. The present obtained results showed that the prepared inactivated EHV-1 adjuvanted with carbomer is free from foreign contaminates; safe in mice and mares and potent in Guinea pigs and mares (table-1), coming in agreement with the directions of OIE (2017). The obtained virus titer after three blind passages in Vero cells was found to be 8.5log10 TCID 50 /ml recording a higher value than that recommended by Hamdy (2016) and OIE (2019). They concluded that EHV-1 titer should not be less than 7log10 TCID 50 /ml before virus inactivation. Such a high virus titer ensures the production of a potent inactivated vaccine.
Insurance of the safety of the presently prepared vaccine showed that all vaccinated horses showed average body temperature and no local or systemic postvaccinal reactions following neither the first of the second dose and there were no abortion in pregnant mares and mice, coming in agreement with the recommendations of OIE (2019).
The preliminary study of the prepared vaccine immune response was conducted in Guinea pigs. Table  (2) showed detectable EHV-1 antibodies with mean ELISA titer 490 and 400 on the third-week postvaccination with carbomer rehydra gel vaccines, respectively. These titers increased to 1019 and 940 by the two vaccines respectively by the second-week post the booster dose (5 weeks post the first dose). These findings coincidence with those of Guo et al. (1989) and Dalia (2017).  Table (4) represented the results of SNT expressed as NI, which was detectable two weeks postvaccination by mean values 0.7 and 0.6 induced by carbomer and rehydra gel with saponin vaccines, respectively. After boosting, there was a gradual increase significantly in NI with mean values 3.5 and 2.8 by the 12 th -week post-vaccination, then began to decline to reach 1.8 and 1.3 by the 28 th week. These reported that NI of EHV-1 was ranged between 1.5-3.5 could resist the challenge of EHV-1 infection. Also, these results agree with Bannai et al. (2014) and OIE (2019), who found that ELISA antibodies begin to increase by day 14 post-vaccination then reached their peak at two months with a 4-fold increase indicated an excellent immune response.
The obtained data indicate that carbomer has a potential effect eliciting higher levels of humoral antibodies with a longer duration of immunity than the conventional Al-hydra gel adjuvant.

CONCLUSION
Depending on the present obtained results, it could be concluded that the prepared EHV-1 inactivated vaccine adjuvanted with carbomer is safe and more potent than that prepared with Al-hydra gel.

Declaration of Competing interest
On behalf of all authors, I hereby declare that no conflict of interest may interfere with the publication of the manuscript.