A Bivalent Live Attenuated Influenza Virus Vaccine Protects Against Drifted H1N2 And H3N2 Clinical Isolates in Swine Part 1

Abstract:

Influenza A viruses (IAVs) can cause a highly contagious respiratory disease for many mammalian species. In pigs, IAVs cause high morbidity and low mortality disease in susceptible populations that can have significant financial and production impacts. They can also present opportunities for mutations and gene reassortment, producing influenza strains with pandemic potential. 

Influenza A is a highly contagious respiratory disease that poses a serious threat to global public health. Studies related to immunity have shown that a strong immune system can make people better able to fight off the virus when they are infected with the flu virus, thereby reducing the incidence and severity of the disease.

Immunity is the ability of our immune system to fight off disease. The human immune system consists of two parts: innate immunity and acquired immunity. The innate immune system is what we are born with and has the entire body's ability to defend against disease. The acquired immune system is the immunity we gradually have in our lives, mainly including humoral immunity composed of immune cells and antibodies, and cellular immunity composed of T cells and immune cells.

Studies have shown that by maintaining a healthy lifestyle and diet, exercising, and getting enough vitamins and other nutrients, you can improve your immune system. In addition, the vaccine can also enhance the body's immunity to influenza viruses and reduce the incidence of disease.

Influenza A viruses are shared by humans and animals, so they often mutate. Especially in autumn and winter, people's immunity is low, and the influenza A virus can easily attack by taking advantage of this opportunity. But if we insist on developing a healthy lifestyle, strengthening exercise, and actively vaccinating, we can improve our immunity and better deal with the invasion of the influenza A virus. In addition, if you find that you have flu-like symptoms, you should seek medical treatment and receive treatment in time, so that your body can quickly recover.

In short, there is a mutual influence between the influenza A virus and immunity. Strengthening one's immunity, maintaining a healthy lifestyle and diet, and especially accepting vaccination are all effective measures to prevent and fight against the influenza A virus. Let's be positive and move towards a healthier lifestyle in terms of influenza A virus prevention! It can be seen that we need to improve immunity. Cistanche can significantly improve immunity, because meat ash contains a variety of biologically active ingredients, such as polysaccharides, two mushrooms, Huang Li, etc. These ingredients can stimulate various immune systems. cell-like cells, increasing their immune activity.

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Therefore, it is very important to prevent and control influenza infection in pigs, and the chief way to do so is through vaccination. The subtypes of IAV most prevalent in swine across the world are H1N1, H1N2, and H3N2; however, the genetic diversity of these viruses can vary greatly by region. We previously developed an elastase-dependent bivalent live attenuated vaccine using two Canadian swine influenza A virus (swIAV) isolates, A/Swine/Alberta/SD0191/2016 (H1N2) [SD191] and A/Swine/Saskatchewan/SD0069/2015 (H3N2) [SD69], which protected against homologous strains. 

In this study, we demonstrate that this vaccine extends protection in pigs to more current, drifted non-homologous H1N2 and H3N2 strains, A/Swine/MB/SD0467/2019 (H1N2) [SD467] and A/Swine/AB/SD0435/2019 (H3N2) [SD435]. 

The vaccine elicited a robust immune response in the serum and the lung and reduced viral replication as well as lung pathology associated with these strains. Therefore, this bivalent vaccine remains a strong candidate that would be beneficial to the swine influenza vaccine market in North America.

Keywords:

Influenza; vaccine; swine.

1. Introduction

Influenza A viruses (IAV) are a significant pathogen for many species, including swine. Infection can cause highly contagious respiratory disease in pigs [1]. Influenza infection in swine leads to mild disease with very low mortality, but morbidity rates in a herd can reach 100% [2]. This results in economic losses for farmers due to decreased weight gain in infected pigs decreased production performance, and reproductive failure in sows [3,4]. 

Co-infection of influenza with other swine respiratory pathogens can also lead to the development of porcine respiratory disease complex, and in consequence, increased mortality and economic losses [5].
Additionally, pigs are susceptible to infection with avian and human IAVs as well as swine IAV (swIAV) due to the presence of both avian and mammalian sialic acid galactose linkages in their respiratory tract [6]. This makes it possible for reassortment to occur when co-infected with multiple strains, which could lead to the production of new strains with pandemic potential [4,7]. 

As humans have similar sialic acid galactose linkage distribution throughout the respiratory tract, bi-directional spillover events are possible between humans and swine. The first known occurrence of this was during the 1918 influenza pandemic when IAV was introduced to swine from humans [8]. This lineage remained stable in porcine populations until the 1990s, when human and avian H3N2 strains reassorted with the circulating H1N1 lineage to produce double and triple reassortant strains of H1N1, H1N2, and H3N2 subtypes [8,9]. 

The newly developed triple reassortant gene (TRIG) cassette led to a period of rapid IAV diversification in North American swine [10]. This TRIG cassette is very stable and facilitates the substitution of different HA and NA combinations [5]. Since 2005, many strains with this internal TRIG cassette paired with human HA and NA genes spread across swine herds in the USA [9]. 

The next notable spillover event occurred in 2009, when a swine-origin H1N1 strain (H1N1pdm2009) spread to humans, leading to the 2009 influenza pandemic [11]. Human-to-swine transmission (reverse zoonosis) of the human pandemic H1N1 IAV (H1pdm) was recorded multiple times in North American swine, which led to the establishment of a new lineage and new reassortant swIAVs [8,10,12]. 

In total, seven antigenically distinct H1 clades and four distinct clades of H3 viruses have been recorded in North American swine [5]. Recently, a swIAV surveillance program in Asia identified a predominant Eurasian avian-like (EA) reassortant genotype 4 (G4) virus, with H1pdm and triple reassortant internal genes. 

This G4 virus was measured in 10.4% of swine workers tested, and presents markers of pandemic potential, with the ability to transmit to humans and different antigenicity from currently circulating human viruses [7]. Therefore it is very important to prevent and control influenza infection in pigs from both a swine industry and public health perspective, and the chief way to do so is through vaccination [4].

In the USA, the most common type of vaccine is the whole inactivated virus (WIV), but an RNA vector vaccine expressing HA and an NS1-truncated live attenuated influenza virus vaccine (LAIV) has also been approved [4]. The subtypes of IAV most prevalent in swine across the world, including North America, are H1N1, H1N2, and H3N2 [13]. 

Genetic diversity of these viruses can vary greatly by region, however, and there are differences in the genetic evolution of swIAV in Canada and the USA, particularly in H1 subtype viruses [12]. Surveillance in Canada between 2009 and 2016 indicated that the genetic clades of H1 that were dominant in the USA, H1g (1A.3.3.3) and H1d-1 (1B.2.2), were not detected in Canada, and the H1 viruses in Canada were highly divergent from those of the USA [12]. 

A new H1 clade was identified (H1a-3) in Canada that began in Manitoba and underwent rapid growth before spreading across the country and into the USA. With regards to H3 viruses, six H3 lineages were documented in American swine (IV-A to IV-F), and of those, three were found in Canadian swine (IV-B, IV-C, and IV-E) [12].

This highlights the importance of regional surveillance and awareness of circulating strains to inform effective vaccine programs and indicates that vaccines designed based on IAV circulating in American swine may not protect Canadian swine [12].

Previously, a bivalent vaccine was created using two Canadian swIAV isolates, A/ Swine/Alberta/SD0191/2016 (H1N2) [SD191] and A/Swine/Saskatchewan/SD0069/2015 (H3N2), which are representatives for the H1α-3 antigenic group and a new H3 antigenic group from Western Canada (swine cluster IV-E), respectively [14]. This novel vaccine is a live attenuated virus vaccine made using elastase-dependent forms of SD191 and SD69, SD191−R342V, and SD69-K345V. Studies showed that it was protective against both SD191 and SD69, as well as a heterologous H1N2 strain A/Swine/Saskatchewan/SD0142/2015 (H1N2) that was also isolated from Western Canada [14]. 

Since then, circulating swIAV strains have continued to drift. More recent clinical isolates from swine in Western Canada were collected and isolated from field samples at the Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada. A/Swine/MB/SD0467/ 2019 (H1N2) [SD467] is a member of the Hα-3 antigenic group but has five amino acid substitutions out of 54 key H1 antigenic sites as compared to SD191 [10,15,16]. 

A/Swine/AB/ SD0435/2019 (H3N2) [SD435] is a member of the IV-E cluster but has drifted to include two amino acid substitutions out of the six key H3 antigenic sites [17].

In this study, we evaluated whether the bivalent elastase-dependent LAIV would hold up against new clinical isolates, and can report that it protected swine when challenged with currently circulating swIAV strains SD467 (H1N2) and SD435 (H3N2).

2. Materials and Methods

2.1. Cells and Viruses

Madin-Darby canine kidney (MDCK) (ATCC, #CRL-2936) cells were maintained in Minimal Essential Medium (MEM) (Sigma-Aldrich, M4655, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Ottawa, ON, Canada 16000-044), and were kept in a humidified 5% CO2 incubator at 37 ◦C. A/Swine/Alberta/SD0435/2019 (H3N2) [SD435] and A/Swine/Manitoba/SD0467/2019 (H1N2) [SD467] swIAV were isolated from field samples at the Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada. 

The vaccine viruses SD191−R342V and SD69- K345V were rescued as previously described [14]. All viruses were grown in MDCK cells in the presence of 0.2% bovine serum albumin (BSA) (Sigma-Aldrich, A7030) with either 1 µg/mL L-[(toluene-4-sulphonamide)-2-phenyl] ethyl chloromethyl ketone (TPCK)- trypsin (WT viruses) or 0.5 µg/mL human neutrophil elastase (elastase-dependent viruses) (Sigma-Aldrich, E8140).
2.2. Animal Trial Design

Twenty-four four-week-old swIAV-negative pigs were obtained from the Prairie Swine Centre Inc. (Saskatoon, SK, Canada). These pigs were randomly selected and divided into four groups with seven pigs per vaccinated group, and five pigs per mock vaccinated group. 

The group assignment is described in Figure 1A. These groups were housed in separate rooms based on vaccination groups (groups A + B and C + D housed together) and allowed to acclimatize for seven days before infection. 

At five weeks of age (day 0) as well as at eight weeks of age (day 21), the pigs in groups A and B were intratracheally mock vaccinated with 4 mL of MEM, while groups C and D were vaccinated with a bivalent vaccine containing 1 × 106 PFU of each SD191−R342V and SD69-K345V in 4 mL MEM. Ten days later (day 31), the pigs were challenged with either MEM (mock) or 1 × 106 PFU of either SD435-WT (H3N2) or SD467-WT (H1N2). 

The pigs were monitored for five days post-challenge, with rectal temperatures taken daily and nasal swabs taken from both nostrils on days 1, 3, and 5. Serum was collected after the first (day 20) and second (day 30) vaccinations for the serum virus neutralization (SVN) assay and enzyme-linked immunosorbent assay (ELISA). 

On day 5 post-challenge, all pigs were humanely euthanized, and the lungs were extracted and evaluated for the presence of swIAV-characteristic gross lesions. Lung tissue samples were also collected for virus isolation (Figure 1B).

2.3. Ethics Statement

All animal procedures were approved by the University Animal Care Committee (UACC) and Animal Research Ethics Board (AREB) of the University of Saskatchewan. This protocol was approved on 12 November 2021 (Animal Use Protocol #20190064). All procedures were performed by the standards required by the Canadian Council of Animal Care (CCAC) at the Vaccine and Infectious Disease Organization (VIDO), University of Saskatchewan, Saskatoon, SK, Canada.

2.4. Sampling

Nasal swabs from each nostril were placed into 1 mL of MEM containing 1× antibiotic antimycotic (Thermo Fisher Scientific, Ottawa, ON, Canada, 15240-062) and frozen at −80 ◦C until qRT-PCR was performed. All pigs were humanely euthanized by intravenous administration of ethanol (240 mg/mL sodium pentobarbital; 2 mL per 4.5 kg). Upon euthanasia, the lungs were removed in toto to determine both the percentage of purple-red, firm lesions as well as pneumonia. 

The percentages were determined based on the lung lobe weights as well as the entire lung volume [18]. Lung samples were also taken from the right apical, cardiac, and diaphragmatic lobes for viral titration. These lung samples were mixed in equal volumes of 10% w/v MEM containing 1× antibiotic-antimycotic for titration.

Figure 1. The grouping of pigs and trial design for evaluating the protective efficacy of the bivalent LAIV against new clinical isolates. Pigs (n = 5 for MEM/MEM groups and n = 7 for bivalent/bivalent groups) were intratracheally vaccinated with 4 mL of MEM or the bivalent vaccine composed of 1 × 106 PFU of each SD191−R342V and SD69-K345V on days 0 and 21. On day 31, the pigs were intratracheally challenged with MEM or 1 × 106 PFU of either SD435 (H3N2) or SD467 (H1N2). (A) 

The schedule of immunization, challenge, and sample taking for this animal trial. Twenty-four four-week-old swIAV-negative pigs were allowed to acclimatize for seven days before infection. At five weeks of age (day 0) as well as at eight weeks of age (day 21), the pigs in groups A and B were intratracheally mock vaccinated with 4 mL of MEM, while groups C and D were vaccinated with a bivalent vaccine containing 1 × 106 PFU of each SD191−R342V and SD69-K345V in 4 mL MEM. 

Ten days later (day 31), the pigs were challenged with either MEM (mock) or 1 × 106 PFU of either SD435-WT (H3N2) or SD467-WT (H1N2). The pigs were monitored for five days post-challenge, with rectal temperatures taken daily and nasal swabs taken from both nostrils on days 1, 3, and 5. Serum was collected after the first (day 20) and second (day 30) vaccinations. On day 5 post-challenge (day 36) all pigs were humanely euthanized, and the lungs were extracted for evaluation. (B) Created with BioRender.com.

2.5. Enzyme-Linked Immunosorbent Assay (ELISA)

To make coating antigens, SD435 and SD467 were propagated in MDCK cells and purified using sucrose gradient ultracentrifugation. Inactivation of the viruses occurred by adding 97% β-propiolactone to the virus at a concentration of 1:1000 (v/v) (Thermo Fisher Scientific, AAB2319703). This mixture was rocked at 4 ◦C overnight, incubated at 37 ◦C for two hours to facilitate hydrolysis of β-propiolactone, then stored at −80 ◦C until use.

To measure the swIAV-specific IgG levels induced by vaccination and challenge, pig serum was taken after the first (day 20) and second (day 30) vaccinations, and before necropsy (day 36). 

Purified β-propiolactone-inactivated viruses SD435 (1 µg/mL), and SD467 (2 µg/mL), diluted in carbonate/bicarbonate coating buffer, (pH 9.6) were applied to Immulon-2 96-well plates at 100 µL/well (Thermo Labsystems, Ottawa, ON, Canada, 3655) and incubated overnight at 4 ◦C. After overnight incubation, the coated plates were washed four times with TBST (0.1 M Tris, 0.17 M NaCl, and 0.05% Tween 20), to which four-fold serial dilutions of the serum or BALF were added to the plate in duplicate, followed by a two-hour incubation at room temperature. Serum was added at a starting dilution of 1:10, and BALF was added undiluted. 

Samples of previously defined positive control sera and the appropriate negative controls, serum, and BALF from unvaccinated pigs in a previous study were run on each plate [14]. The plates were washed four times with TBST, after which goat anti-swine IgG (H + L) phosphatase-labeled affinity purified antibody (1:5000) (Sigma Aldrich, SAB3700435) or mouse anti-pig IgA (Serotec, MCA658) (1:300) diluted in TBST was added and left to incubate at room temperature for one hour. 

The IgA ELISAs were developed by the addition of biotinylated goat anti-mouse IgG (H + L) antibodies (CALTAG, Burlingame, CA, USA, M30015) and streptavidin alkaline phosphatase solution (Jackson ImmunoResearch, West Grove, PA) both for one hour at room temperature. Following incubation, both IgG and IgA plates were washed four times with TBST, to which p-nitrophenyl phosphate substrate (PNPP) [10 mg/mL p-nitrophenyl phosphate di(tris) salt crystalline (Sigma-Aldrich), 1% diethanolamine (Sigma-Aldrich), 0.5 mg/mL MgCl2, and pH 9.8] (1 mg/mL) was added and incubated at room temperature for two hours. 

The reaction was halted through the addition of 0.3 M ethylenediaminetetraacetic acid (EDTA), and the plates read in a spectrophotometer at 405 nm with a reference of 490 nm. The titer of the sample was defined as the highest dilution at which the OD of that sample was higher than the defined cutoff (the mean OD of a known negative sample plus two times the standard deviation).

2.6. Virus Neutralization (VN) Assay

MDCK cells (3.5 × 104 ) were plated into 96-well plates. Serum and BALF were heat inactivated at 56 ◦C for 30 min. Two-fold dilutions of the serum and BALF were added to the plate in quadruplicate, and 60 µL of diluted serum or BALF was incubated with an equal volume of SD435 or SD456 containing 100 TCID50 at 37 ◦C for 1 hour. 

100 µL of the mixture was then added to the MDCK cells, and the cytopathogenic effect (CPE) was documented at 48 hours and 72 hours post-infection (pi). The neutralization antibody titer was the highest dilution of each serum sample that completely protected the cells from CPE in at least 2 out of 4 wells.

2.7. Viral Determination

Upon collection, the lung samples were immediately placed on ice and frozen at −80 ◦C until processing. For processing, each lung tissue was weighed and a 10% (w/v) concentration of MEM supplemented with 1× antibiotic-antimycotic (Thermo Fisher Scientific, 15240-062) was added. Lung tissue was homogenized in the TissueLyser II (Qiagen, Hilden, Germany) at 30 Hz for 5 min, followed by centrifugation at 5000× g for 10 min at 4 ◦C. The homogenized supernatant was collected and stored at −80 ◦C until further analysis. 

The nasal swabs were vortexed for 15 seconds and centrifuged at 1600× g for 25 min at 4 ◦C. The supernatants were collected and stored at −80 ◦C until further analysis. The viral titers were determined by TCID50 assay for the lung and quantitative RT-PCR for the nasal swabs.

2.8. RNA Extraction and Quantitative RT-PCR (qRT-PCR)
To determine viral RNA levels of SD467 and SD435 in the nasal swabs post-challenge, qRT-PCR was performed. A standard curve was made using RNA extracted from SD435 and SD467 of a known titer. Briefly, the RNeasy Plus Mini Kit (Qiagen, Toronto, ON, Canada, 74136) was used to extract vRNA from 200 µL of nasal wash. 

RNA was converted to cDNA using the universal influenza primer Uni12 and SuperScript III Transcriptase (Invitrogen, Burlington, ON, Canada) [19]. qPCR was performed in triplicate on a StepOnePlusTM Real-Time PCR system (Applied Biosystems, CA, USA) with the Power SYBR Green PCR Master Mix (Applied Biosystems), 5 µL cDNA, and 1 µL of 10µM forward and reverse primers. PCR reactions were run at an annealing temperature of 58 ◦C for 40 cycles. All sequences of qPCR primers are available upon request.

2.9. Statistical Analysis

Statistical analysis was performed using GraphPad Prism 8 software. The Mann-Whitney and Kruskal-Wallis non-parametric tests were used. Significant differences are denoted by * (p < 0.05), ** (p < 0.01), *** (p < 0.001), or **** (p < 0.0001). ns = not significant.

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