Elsevier

Vaccine

Volume 30, Issue 2, 5 January 2012, Pages 280-288
Vaccine

Vaccination with NS1-truncated H3N2 swine influenza virus primes T cells and confers cross-protection against an H1N1 heterosubtypic challenge in pigs

https://doi.org/10.1016/j.vaccine.2011.10.098Get rights and content

Abstract

The diversity of contemporary swine influenza virus (SIV) strains impedes effective immunization of swine herds. Mucosally delivered, attenuated virus vaccines are one approach with potential to provide broad cross-protection. Reverse genetics-derived H3N2 SIV virus with truncated NS1 (NS1Δ126 TX98) is attenuated and immunogenic when delivered intranasally in young pigs. We analyzed T-cell priming and cross-protective efficacy in weanling piglets after intranasal inoculation with NS1Δ126 TX98 versus wild type TX98. In vivo replication of the truncation mutant was minimal compared to the wild type virus. T-cell responses were greater in magnitude in pigs infected with the wild type virus in in vitro restimulation assays. According to the expression of activation marker CD25, peripheral T cell recall responses in NS1Δ126 TX98 infected pigs were minimal. However, intracellular IFN-γ data indicate that the attenuated virus induced virus-specific CD4+CD8, CD4+CD8+, CD4CD8+, and γδ T cells within 28 days. The IFN-γ response appeared to contract, as responses were reduced at later time points prior to challenge. CD4+CD8+ cells isolated 5 days after heterosubtypic H1N1 challenge (day 70 overall) showed an elevated CD25 response to virus restimulation. Pigs previously infected with wild type TX98 were protected from replication of the H1N1 challenge virus. Vaccination with NS1Δ126 TX98 was associated with significantly lower levels of Th1-associated cytokines in infected lungs but provided partial cross-protection against the H1N1 challenge. These results demonstrate that NS1Δ SIV vaccines can elicit cell-mediated cross-protection against antigenically divergent strains.

Highlights

► Attenuated H3N2 influenza vaccine with truncated NS1 was administered to pigs. ► Vaccine induced broadly reactive mucosal antibodies. ► Vaccine primed T cells which were elevated in peripheral blood after infection. ► Vaccinated animals had reduced shedding of heterosubtypic H1N1 challenge virus.

Introduction

Pigs have been recognized as a natural host of influenza A virus since the virus was first isolated in 1930. After a relatively slow evolution of classical H1N1 swine influenza virus (SIV) in the North American swine population, a new reassortant H3N2 lineage emerged and was established around 1998 [1]. Compared to the classical swine H1N1, this SIV lineage has shown a propensity for frequent gene reassortment and rapid antigenic drift [2], [3], [4], [5]. Variants of this reassortant lineage, which have been isolated routinely in recent years, possess H3N2, H1N1, and H1N2 serotypes (reviewed by Vincent et al. [6]). Segments of the “triple reassortant internal gene” (TRIG) cassette were acquired from swine, avian, and human influenza A lineages [1]. Occasional reassortants have emerged in swine with novel surface glycoproteins with the TRIG [5], [7]. Five of the six gene segments which encode internal proteins in the 2009 pandemic H1N1 virus were derived from this North American TRIG lineage [8]. The emergence of this virus illustrates the risk to public health as influenza A virus genes of diverse origins are shuffled together in the backbone of the mammalian-adapted triple reassortant virus.

Additional antigenically distinct reassortant viruses could potentially emerge from swine lineages into the human population [9]. New variants may also have great economic costs to the swine industry. Therefore, there is a strong rationale for investigations into the immunological relationships among recent and emerging subtypes or genotypes of swine-origin reassortant influenza viruses. Experimental evaluation of modern vaccine technology for SIV in the swine host is important for achieving greater control of the various strains in swine populations and limiting the risk of transmission to humans. The antigenic diversity of influenza viruses circulating and becoming established in global swine populations suggests that veterinary vaccination programs are unlikely to produce neutralizing humoral immunity against all emerging strains. Therefore, an important consideration in evaluating candidate vaccines should be the degree to which they elicit cross-reactive cell-mediated immunity. Although influenza-specific T cells do not provide sterilizing immunity, they protect more broadly than neutralizing antibodies against heterologous and heterosubtypic strains through viral clearance [10], [11]. Broadly cross-reactive T cell epitopes are typically located in internal proteins of influenza viruses such as the nucleoprotein (NP) and matrix (M1) segments, which are more conserved than the surface hemagglutinin (HA) and NA glycoproteins [12], [13], [14]. Cellular immunity to influenza in the absence of cross-reactive antibody can markedly reduce viral replication in humans and pigs [15], [16].

Live virus vaccines are considered to be more effective than inactivated or non-replicating vaccines as inducers of cellular immunity, particularly for MHC class I-restricted T cells, but all licensed SIV vaccines in the US are based on inactivated virus antigens. Molecular approaches have been used to construct mutated SIV genomes which confer attenuated replication properties, including reduced NS1 suppression of type I interferon, dependence on the enzyme elastase for HA cleavage, and temperature-sensitive mutations in polymerase genes [17], [18], [19]. Truncation of NS1 protein in triple reassortant H3N2 strain A/Sw/Texas/4199-2/98, from a length of 230 amino acids to 126, produced a mutant with restricted replication in the swine respiratory tract but strong immunogenic properties [20]. Intranasal inoculation of pigs with this virus (NS1Δ126 TX98) resulted in robust protection against homologous challenge and significantly reduced viral replication and clinical signs upon challenge with a drift variant strain [21]. One likely factor in the partial heterologous protection was the production of mucosal IgA antibodies that had significant cross-reactivity against the drift variant. No investigation was made into the priming of T cells subsets in the NS1Δ126 TX98 immunized pigs, but we hypothesize that cross-protection was mediated at least in part by the cell-mediated immune (CMI) response. A theoretical concern with live SIV vaccines, such as NS1Δ126 TX98, is the possibility that they would undergo reassortment with circulating strains and produce variants with altered virulence, transmissibility, or host range. This vaccine does not possess any novel genetic elements to contribute to the current circulating pool of influenza viruses as the parental H3N2 TRIG background from which the NS1Δ126 TX98 vaccine strain was generated continues to circulate widely in the US swine population, and therefore concerns about the swine host acting as a mixing vessel in this context should be minimal [6]. Attenuated H1N1 SIVs made by introducing elastase dependent mutations also protected pigs against homologous and heterologous H1N1 challenge while eliciting significant T-cell responses and lung IgA titers against both strains [22].

In the present investigation we analyzed the CMI response following NS1Δ126 TX98 or wild type virus vaccination by measuring ex vivo responses of each major T cell subset from vaccinated or unvaccinated pigs to attenuated or wild-type virus. Serum and peripheral blood mononuclear cells (PBMC) were collected at multiple time points following vaccination, the last of which was 5 days after heterologous H1N1 virus challenge. The heterologous challenge virus was a reassortant H1N1 (rH1N1) subtype isolated after classical swine H1N1 surface glycoproteins reassorted with H3N2 SIV [23]. The heterosubtypic challenge strain carries the TRIG cassette, which is common to the TX98-derived vaccine candidates (Supplemental Table 1). Immunization with either attenuated or wild type virus elicited antigen-specific responses by peripheral T cells. After heterologous challenge of the immunized animals, T-cell sensitivity to viral stimulation was augmented again in both groups, but the phenotypic characteristics of responding cells were shifted markedly.

Section snippets

Viruses

The H3N2 isolate A/swine/Texas/4199-2/1998 (TX98) was propagated in allantoic cavities of 10-day old embryonated chicken eggs to produce an inoculum for vaccination. To limit recall responses to non-viral endogenous egg antigens, the IA04 H1N1 challenge viral stock was propagated in Madin-Darby canine kidney (MDCK) cells. The same strains were grown in MDCK cell cultures to generate recall antigen for ex vivo stimulation of T cells. Live influenza A virus NS1Δ126 TX98 was generated by reverse

Vaccine virus replication in naïve pigs

TX98 mutants with truncated NS1 replicated much less extensively in the upper respiratory tract than wild type TX98 (Fig. 1). The wild type virus, administered intranasally, replicated sufficiently to produce nasal swab viral titers of approximately 104 TCID50/ml through day 4. The mean titer dropped below 10 TCID50/ml on day 6. In pigs vaccinated with the attenuated NS1Δ126 TX98 mutant, the mean viral titers did not exceed 10 TCID50/ml on days 2 or 4, and no virus was detected on day 6. This

Discussion

Multiple molecular strategies have been developed in recent years for the design of live-attenuated influenza A vaccine candidates. NS1 deletion or truncation attenuates an influenza virus by reducing its ability to antagonize the type I IFN response [28]. It was hypothesized that this class of mutant viruses would elicit robust adaptive immune responses, despite attenuated replication, because restraints on the early innate response would be relaxed. Indeed, viruses with deleted or truncated

Acknowledgements

Authors wish to thank Michelle Harland, Deborah Adolphson, Ann Vorwald, Sarah Pohl, Dr. Laura Miller, Dr. Janice Ciacci-Zanella, and Dr. Eraldo Zanella for technical assistance and would also like to also thank Jason Huegel and Brian Pottebaum for animal caretaker assistance. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. Funding was

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