Recombinant equine herpesvirus 1 (EHV-1) vaccine protects pigs against challenge with influenza A(H1N1)pmd09
Highlights
► Equine herpesvirus type 1 robustly expresses hemagglutinin of pandemic H1N1 (“swine influenza virus”). ► Pigs are clinically protected against challenge infection after vaccination. ► Vaccine was utilized with no purification or adjuvant. ► Viral loads are reduced in vaccinated versus non-vaccinated animals.
Introduction
Swine influenza (SI) is a highly contagious viral infection in pigs and is characterized by coughing, nasal discharge, elevated temperatures, breathing difficulties and reduced appetite (Amorij et al., 2010, Tang et al., 2002, Wang and Palese, 2009b). The disease is caused by SIV, which belongs to the influenza A virus genus in the Orthomyxoviridae family. The influenza A viruses are characterized by a segmented genome of eight single-stranded RNA molecules of negative polarity (Castrucci et al., 1994, Palese and Shaw, 2007, Webster and Bean, 1978). Influenza A viruses undergo antigenic shift (or reassortment) and drift processes leading to the continued generation of new virus variants. Pigs have both major types of viral receptors in the respiratory tract that are characterized by sialic acids with α2,3 and α2,6 linkages, respectively (Ito et al., 1998, Ito et al., 2000). Therefore, pigs have been considered an important reservoir host or even a mixing vessel for the generation of new reassorted strains with pandemic capacity (Castrucci et al., 1994, Garten et al., 2009, Kida et al., 1994, Schultz et al., 1991).
In April 2009, a novel swine-origin H1N1 influenza A virus, later by convention referred to as A(H1N1)pdm09 (WHO, 2011), was identified. The virus has a distinct combination of gene segments from both North American and Eurasian swine influenza lineages as well as avian and human lineages (Furuse et al., 2010, Garten et al., 2009, Girard et al., 2010, Smith et al., 2009). The susceptibility of pigs to this particular virus strain was confirmed in several experimental studies (Lange et al., 2009, Vincent et al., 2010b). The clinical signs associated with A(H1N1)pdm09 infection in pigs were similar to those caused by endemic swine influenza strains. As a consequence of the appearance of A(H1N1)pdm09, the porcine industry was severely impacted as pork consumption dropped primarily because of the misconception that the disease can be transmitted through meat (Pappaioanou and Gramer, 2010).
Infected pigs may, however, become a source of infection for humans, even if the virus does not succeed in becoming endemic in pig populations (Irvine and Brown, 2009, Van Reeth et al., 2007). Therefore, vaccination of pig populations by highly effective vaccines that are able to reduce virus excretion may help reduce the risk for human infection. The viral hemagglutinin (HA), one of the two envelope glycoproteins, is the main viral antigen (Hessel et al., 2011, Hghihghi et al., 2011). A number of studies have demonstrated that immunization with recombinant HA is capable of inducing both cell-mediated and humoral immune responses (Hessel et al., 2011). Currently available commercial swine influenza vaccines are traditional, inactivated whole virus preparations containing the H3N2 and H1N1 subtypes. The inactivated virus vaccines are produced either in eggs or in cell culture and usually efficacious at reducing clinical signs, although it was shown that they may enhance disease in some cases (Kobinger et al., 2010, Vincent et al., 2008). Previous studies suggested that cell-mediated and/or mucosal responses, which are not stimulated by inactivated virus vaccines, are essential for induction of heterologous immunity (Ma and Richt, 2010, Reeth et al., 2004). Moreover, the use of whole virus vaccines does not allow for differentiation between infected and vaccinated animals. Pigs are often asymptomatic after SIV infection but can still spread the virus. For these reasons, efforts have concentrated on the development of alternative vaccine production methods by engineering recombinant virus vaccines against SIV (Sipo et al., 2011, Tang et al., 2002, Tian et al., 2006, Wesley et al., 2004). In our study, we used equine herpesvirus type 1 (EHV-1) as a delivery vector for influenza virus HA to protect pigs against challenge infection with the A(H1N1)pdm09. EHV-1 is a member of the genus Varicellovirus, which belongs to the subfamily Alphaherpesvirinae in the Herpesviridae family. The EHV-1 genome is a double-stranded DNA of 150-Kbp in length (Davison et al., 2009). The virus is highly prevalent in the horse population and causes mild to severe clinical disease that includes respiratory distress, abortion storms and neurological disorders in equines (Allen and Bryans, 1986, Telford et al., 1992). The potential of the EHV-1 RacH modified live virus (MLV) vaccine strain as an immunization vector has been highlighted by its ability to stably and efficiently deliver immunogenic proteins and induce both humoral and cellular immune responses (Osterrieder et al., 1996, Rosas et al., 2007a, Rosas et al., 2007b, Rosas et al., 2008). The RacH strain has a proven safety record in horses and in a number of other animal species including mice, dogs and cattle (Rosas et al., 2007a, Rosas et al., 2007b, Rosas et al., 2008, Said et al., 2011). Its attenuation could be attributed to a deletion of both copies of gene 67. Other genomic alterations, such as the truncation of the glycoprotein B, also contribute to its complete a pathogenicity for various species (Hübert et al., 1996, Neubauer et al., 1999, Osterrieder et al., 1996).
We previously reported on the construction of an EHV-1-based vaccine expressing the H1 derived from A(H1N1)pdm09 A/California/4/2009 that was termed rH_H1 (Said et al., 2011). We demonstrated that the rH_H1 vaccine was able to induce an immune response in mice in which a reduction of clinical signs and faster virus clearance from the respiratory tract was documented. Here, we evaluate the extent to which the vaccine is able to protect pigs against challenge infection with the A(H1N1)pdm09 virus. We show that vaccinated pigs mounted robust immune responses and had significantly reduced virus loads in the respiratory tract after challenge infection.
Section snippets
Viruses and cells
Rabbit kidney (RK13) cells were maintained in Earle's minimal essential medium (EMEM) supplemented with 5% heat-inactivated fetal bovine serum (FBS) and antibiotics (100 U/ml penicillin and 0.1 mg/ml streptomycin). Modified live EHV-1 expressing H1 (rH_H1) of A(H1N1)pdm09 (A/California/4/2009) was propagated in RK13 cells (Said et al., 2011). For challenge infection, A/Bayern/74/2009/H1N1 (kindly provided by B. Schweiger, Robert Koch-Institut, Berlin, Germany) originating from a human patient was
Statistical analysis
Student's t-test was used to compare ELISA NP and HI titers in serum samples as well as viral loads in nasal swabs in rH_H1- and mock-immunized groups. The significance level was set at 0.05.
Serological responses are induced in rH_H1-immunized pigs prior to experimental challenge
Serological studies were done in piglets to determine whether rH_H1 was capable of inducing humoral immune responses against A(H1N1)pmd09 and heterologous H1 strains. Serum samples were examined by HI after the second vaccination, which showed that all animals vaccinated with rH_H1 mounted high antibody titers against A(H1N1)pmd09 (Fig. 1). As expected, all animals in the mock-immunized group did not show any H1 specific antibody Moreover, anti-influenza virus NP antibody levels were determined
Discussion
Pandemic influenza A(H1N1)pdm09, antigenically and genetically divergent from seasonal H1N1, caused a flu pandemic in humans starting in 2009. The development of an effective vaccine to limit transmission of A(H1N1)pdm09 in animal reservoir hosts and from reservoir hosts to humans and other animals is necessary. Preparation of inactivated influenza vaccines is labor-intensive, may not protect for long periods, and usually requires repeated immunizations to induce protective immunity in swine.
Acknowledgments
This work was supported by the U.S. Department of Homeland Security under Grant Award Number 2010-ST-AG0001 and unrestricted funds from the Freie Universität Berlin to N.O. A.S. was supported by a grant from the Egyptian Ministry of Higher Education.
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