A lipidated form of the extracellular domain of influenza M2 protein as a self-adjuvanting vaccine candidate

Highlights

• Lipopeptides based on the extracellular domain of influenza M2 protein constructed.

• Immunization of the lipopeptides protects mice against lethal viral challenge.

• Higher IgA and IgG2b antibodies in BAL by the i.n. immunization than the s.c. route.

• i.n. immunization protects mice better against viral challenge than by s.c. route.

• Viral challenge boosts anti-M2e antibody specificity introduced by M2e immunization.

Abstract

The highly conserved extracellular domain of Matrix protein 2 (M2e) of influenza A virus has been previously investigated as a potential target for an universal influenza vaccine. In this study we prepared four lipopeptide influenza vaccine candidates in which the TLR2 agonist S-[2,3-bis(palmitoyloxy)propyl] cysteine, (Pam2Cys) was attached to either the N- or C-terminus of the M2e consensus sequence SLLTEVETPIRNEWGCRCNDSSDP and its analogue sequence with the two cysteine residues replaced with serine residues. The results of animal study show that each of these lipopeptides induced strong M2e-specific antibody responses in the absence of extraneous T helper cell epitope(s) which are normally incorporated in the previous studies or addition of extraneous adjuvant and that these antibodies are protective against lethal challenge with influenza virus. Comparison of different routes of inoculation demonstrated that intranasal administration of M2e lipopeptide induced higher titers of IgA and IgG2b antibodies in the bronchoalveolar lavage than did subcutaneous vaccination and was better at mitigating the severity of viral challenge. Finally, we show that anti-M2e antibody specificities absent from the antibody repertoire elicited by a commercially available influenza vaccine and by virus infection can be introduced by immunization with M2e-lipopeptide and boosted by viral challenge. Immunization with this lipidated form of the M2e epitope therefore offers a means of using a widely conserved epitope to generate protective antibodies which are not otherwise induced.

Introduction

Immunization against influenza remains the most effective way of preventing influenza which nevertheless remains a significant cause of morbidity and mortality worldwide [1]. Conventional influenza vaccines are manufactured as a cold adapted live influenza virus [2] or as “split” virus or purified viral protein preparations [3]. These vaccines provide protection against influenza infection principally by inducing neutralizing antibodies directed against the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) of the influenza virus [3]. Because the antigenic properties of HA and NA change seasonally through the processes of antigenic drift, which is driven by selection pressure from the adaptive immune response, vaccines need to be reformulated on a yearly basis. When two or more strains of virus combine to form a new subtype by the process of antigenic shift existing vaccines are ineffective and the population at large lacks immunity [3]. In contrast, vaccines based on conserved viral proteins could potentially provide cross-reactive protection against strains of diverse influenza virus subtypes and therefore would not require annual update of vaccine formulation making these suitable for stockpiling as an immediate defense should a pandemic occur [4], [5].

The extracellular domain of the matrix protein 2 of influenza A virus (M2e) is highly conserved across all known human influenza A viruses with little change over the last 80 years [6]. It is this property that has resulted in M2e being targeted as a “universal” vaccine [7], [8], [9], [10], [11]. Many different M2e-based constructs have been explored as vaccine candidates; these include M2e epitope-carrier conjugates [12], [13], M2e fusion proteins [14], [15], [16], [17], [18], multiple M2e antigenic peptides [19], [20], [21], [22], and M2 DNA [9], [23]. Most of these studies have demonstrated protection against challenge with homologous or hetero-subtypic viruses in mice [10], [11], ferrets [12], [23] or in rhesus monkeys [12], [24].

The majority of these studies have utilized vaccine constructs in which M2e is fused genetically or chemically to a carrier protein or peptide as a source of T cell help [19], [20], [21], [22], [25], [26], [27], [28], [29], [30]. Previously, we [30] and others [31] showed that M2e alone could induce M2e-specific antibody responses in BALB/c mice when administered with Freund's adjuvant, results which indicate that there is an active T helper cell epitope(s) for BALB/c mice within the M2e sequence.

In this study we examined the immunogenic properties of the M2e peptide alone in the absence of carrier protein, T helper cell epitope or extraneously added adjuvants. We made four lipopeptide constructs by incorporating the TLR2 agonist S-[2,3-bis(palmitoyloxy)propyl] cysteine (Pam2Cys) to either the N-terminus or the C-terminus of the M2e consensus sequence SLLTEVETPIRNEWGCRCNDSSDP or to the M2e analogue sequence with the two cysteine residues replaced with serine residues. We investigated the immunogenicity of these lipopeptides in mice and their ability to protect against lethal influenza virus challenge. The anti-M2e antibody response following viral infection in M2e-immunized animals was also studied.