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. Therefore, it is very important to prevent and control influenza infection in pigs, and the chief way to do so is through vaccination. 

Influenza A virus is a common respiratory infectious disease, which is highly contagious and susceptible to a wide range of people. Especially for people with weakened immune systems, influenza A viruses can cause more serious health problems. Therefore, it is very important to maintain sufficient immunity to prevent influenza A virus infection.

Our body's immune system is the first line of defense against viruses and bacteria from invading the body. The basic function of the immune system is to resist the invasion of pathogens through white blood cells, lymphocytes, and antibodies, to maintain the health of the body. Therefore, good immune function is an important protective measure to protect us from various infectious diseases.

At the same time, some healthy living habits can also help strengthen immunity. Such as maintaining adequate sleep, moderate exercise, a balanced diet, reducing stress, and so on. These living habits can promote the production of hormones and immune factors in the body, making our immune system stronger.

In addition to your positive actions, you can also increase your body's immunity by getting vaccinated. At present, there is a vaccine against influenza A virus, which can effectively prevent influenza virus infection and reduce the risk of disease through vaccination. Especially for susceptible groups such as the elderly, children, and pregnant women, more attention should be paid to vaccination.

It should be noted that our body's immunity is not static but changes with factors such as age and environment. Therefore, we need to always pay attention and adjust our lifestyle to maintain good immunity.

In summary, adequate immunity is an important means to prevent influenza A virus infection. Through adjustments to health regimens such as sleep, exercise, and diet, as well as vaccinations, we can make our bodies healthier and more resistant to virus attacks. It can be seen that we need to improve immunity. Cistanche can significantly improve immunity. The polysaccharides in meat can regulate the immune response of the human immune system, improve the stress ability of immune cells, and enhance the bactericidal effect of immune cells.

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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.

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