Inclusion of the murine IgGκ signal peptide increases the cellular immunogenicity of a simian adenoviral vectored Plasmodium vivax multistage vaccine

Highlights

• The murine IgGκ signal peptide was included in the Simian Adenovirus 36 vector.

• A P. vivax multi-stage vaccine candidate was encoded downstream of the SP.

• Mice were immunized with the unmodified or SP-SAd36 vector, and two protein boosts.

• SP-SAd36 immunization induced higher antibody avidity.

• SP-SAd36 immunization induced more IFN-γ and IL-2-secreting CD4 T cells.

Abstract

Introduction

Cellular and humoral immune responses are both involved in protection against Plasmodium infections. The only malaria vaccine available, RTS,S, primarily induces short-lived antibodies and targets only a pre-erythrocytic stage antigen. Inclusion of erythrocytic stage targets and enhancing cellular immunogenicity are likely necessary for developing an effective second-generation malaria vaccine. Adenovirus vectors have been used to improve the immunogenicity of protein-based vaccines. However, the clinical assessment of adenoviral-vectored malaria vaccines candidates has shown the induction of robust Plasmodium-specific CD8+ but not CD4+ T cells. Signal peptides (SP) have been used to enhance the immunogenicity of DNA vaccines, but have not been tested in viral vector vaccine platforms.

Objectives

The objective of this study was to determine if the addition of the SP derived from the murine IgGκ light chain within a recombinant adenovirus vector encoding a multistage P. vivax vaccine candidate could improve the CD4+ T cell response.

Methods

In this proof-of-concept study, we immunized CB6F1/J mice with either the recombinant simian adenovirus 36 vector containing the SP (SP-SAd36) upstream from a transgene encoding a chimeric P. vivax multistage protein or the same SAd36 vector without the SP. Mice were subsequently boosted twice with the corresponding recombinant proteins emulsified in Montanide ISA 51 VG. Immunogenicity was assessed by measurement of antibody quantity and quality, and cytokine production by T cells after the final immunization.

Results

The SP-SAd36 immunization regimen induced significantly higher antibody avidity against the chimeric P. vivax proteins tested and higher frequencies of IFN-γ and IL-2 CD4+ and CD8+ secreting T cells, when compared to the unmodified SAd36 vector.

Conclusions

The addition of the murine IgGκ signal peptide significantly enhances the immunogenicity of a SAd36 vectored P. vivax multi-stage vaccine candidate in mice. The potential of this approach to improve upon existing viral vector vaccine platforms warrants further investigation.

Introduction

The life cycle of Plasmodium parasites is known for its complexity, and as a result, immunity to malaria infections in vertebrates relies on both humoral and cellular immune responses. Early passive transfer experiments demonstrated the protective role of IgG antibodies derived from malaria immune adults when used as a therapeutic intervention [1]. Clinical trials of sporozoite inoculation have revealed that IFN-γ producing T cells are associated with protection against malaria [2]. Based on this evidence, a multistage vaccine capable of eliciting both cytophilic antibodies and antigen-specific T cells would likely enhance the protective efficacy of a comprehensive vaccination strategy.

The RTS,S/AS01 vaccine represents a significant breakthrough as the first P. falciparum malaria vaccine that has completed Phase 3 clinical trials [3]. However, RTS,S has reported low efficacy due in part to protection based primarily on antibodies against the circumsporozoite protein (CSP) central repeat region [4] present in pre-erythrocytic stage forms, which wane rapidly and require boosting immunizations to maintain efficacy [5]. The inclusion of erythrocytic stage targets to control parasites that evade liver clearance and enhancing cellular immunogenicity are likely necessary for developing an effective second generation of malaria vaccines.

Adenoviral vectored malaria vaccines have been able to improve the immunogenicity of protein-based vaccines [6], [7], [8], [9] and induce protective Plasmodium-specific CD8+ T cells in pre-clinical and clinical studies [10], [11], [12], but low induction of CD4+ T cell suggests further improvements to adenoviral vectors should be investigated [11]. Recent studies examining the induction of CD4+ T cells following vaccination with an Ad5 vector have shown that significantly lower frequencies of antigen-specific CD4+ T cells are induced when compared to acute infection, an effect that could be attributed to lower IL-2 signaling [13]. Increasing secretion or altering post-translational modifications of adenoviral transgene products might result in improved presentation of vaccine antigens to CD4+ T cells.

Signal peptides (SP), also referred to as signal sequences, are short peptides (∼20–30 residues) that can influence the targeting pathway of the protein and promote protein secretion or specific post-translational modifications such as glycosylation [14]. As a result, SP from highly secreted proteins have been used to improve protein secretion levels of recombinant proteins in cell lines [15], [16], [17], as well as for ectopic expression of endogenous adenoviral genes [18]. Recently, the inclusion of an SP into a DNA vaccine targeting HPV oncogenes was found to induce potent cellular and humoral immune responses that protected against tumor challenge [19]. Of the signal peptides used to improve transgene expression, the sequence derived from the murine immunoglobulin kappa (IgGκ) light chain (METDTLLLWVLLLWVPGSTG), is one of the most well characterized [15], [16], [17].

We hypothesized that the addition of the signal peptide derived from murine IgGκ light chain upstream of a transgene delivered via a recombinant adenovirus vector would improve the CD4+ T cell response to the transgene product in comparison to vaccination with the same recombinant vector without the signal peptide [10]. Here we demonstrate that the addition of the murine IgGκ SP improves the immunogenicity of an adenoviral vectored P. vivax multistage vaccine [20], [21] in mice by significantly increasing IFN-γ and IL-2 secretion by CD4+ T cells, and improving antibody avidity. To our knowledge, this is the first report of the insertion of a signal peptide sequence as part of an adenoviral transgene with the goal of improving the immunogenicity of an adenoviral vectored vaccine candidate.