Efficient protein boosting after plasmid DNA or recombinant adenovirus immunization with HIV-1 vaccine constructs

Abstract

DNA plasmids and recombinant adenovirus serotype-5 (rAd5) vectors are being studied in human clinical trials as HIV-1 vaccine candidates. Each elicits robust T-cell responses and modest antibody levels. Since protein immunization alone elicits antibody but not CD8 T-cell responses, we studied protein boosting of DNA and rAd5 HIV-1 vaccine vectors. A single Env protein immunization provided a marked boost in antibody titer in guinea pigs primed with either DNA or rAd5 vaccines, and the resulting antibody binding and neutralization levels were similar to those attained after thee sequential protein immunizations. Since both T-cell immunity and neutralizing antibodies are thought to be required for protection against HIV-1, it may be possible to establish a balanced T-cell and antibody response with appropriate vectored vaccines and improve the neutralizing antibody titer with protein boosting.

Introduction

The continued spread of the human immunodeficiency virus type 1 (HIV-1) worldwide highlights the need for an effective vaccine. Based on studies of HIV-1 infection in humans, and SIV infection of macaques, it is believed that CD8 T-cells play an important role in control of viral replication [1]. It is hoped that vaccine elicited T-cell responses will be able to limit viral replication and prevent HIV-1 associated disease progression in those individuals who become infected. However, antibodies may be required to prevent the acquisition of HIV-1 infection. Neutralizing antibodies can block viral entry into human T-cells in vitro and can protect non-human primates from a chimeric simian-human immunodeficiency virus (SHIV) infection [2], [3]. Thus, a major goal is to develop an HIV-1 vaccine strategy that will generate both cellular immunity and high titers of virus neutralizing antibodies. Serial immunization with HIV-1 envelope (Env) protein immunogens can result in high antibody levels, but little or no CD8 T-cell responses [4], [5], [6], [7], [8], [9]. One approach to generating both cellular immunity and neutralizing antibodies is to initiate immunization with a gene based vector that is known to elicit CD4 and CD8 responses, and boost with a protein immunogen.

Current gene-based immunogens, such as plasmid DNA and rAd5, have been widely studied in animal models [10], [11], [12], [13], [14], [15], and have entered phase I and II human testing [16], [17], [18], [19]. In addition, numerous groups have utilized vector prime, protein boost regimens to improve the potency or durability of the immune response to HIV or SIV antigens [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30]. In this study, we evaluated the ability of our existing clinical vaccine products, multi-gene plasmid DNA and rAd5, to prime for a booster immunization with a soluble oligomeric Env protein. The plasmid DNA and rAd5 vectors used here were the same as those currently being studied in human trials [18], [19]. The DNA product consists of six individual plasmids. Three encode gp145 Env proteins (clade A–C); the remaining three encode Gag, Pol and Nef. The rAd5 product contains four separate rAd5 vectors. Three encode gp140 versions of the same Env proteins as above; a fourth encodes a Gag-Pol fusion protein. Data from non-human primate studies show that a series of three DNA immunizations, or a single rAd5 immunization, generates robust antigen specific CD4 and CD8 responses and moderate antibody levels [31], [32]. Recent phase I studies in humans have confirmed these results [18], [19]. Additionally, the combination of DNA prime, rAd5 boost has been extensively studied in small animals and non-human primates, and results in robust cellular immunity and high antibody levels [13], [32], [33], [34], [35], [36], [37]. However, we have not systematically studied the ability of these DNA or rAd5 vaccine candidates to prime for a protein boost. Thus, these studies focus on the anti-Env response elicited by DNA or rAd5, and boosted with a soluble Env protein.

A soluble oligomeric gp140 Env protein based on the SF162 strain, and containing a deletion of the second variable region (V2), has been studied in animal models and is currently being tested in phase I human trials. Immunization with the dV2gp140 protein generates high titer antibody levels including homologous neutralizing antibodies [6], [38], [39], [40], [41]. In addition, dV2gp140 elicits high antibody titers in monkeys primed with a DNA plasmids encoding the homologous env gene [42], [43]. To further asses the potential for protein boosting of both DNA and rAd5, we formulated the dV2gp140 Env protein with the MF59 adjuvant alone, or with one of two different classes of a synthetic oligodeoxynucleotide (ODN) containing unmethylated deoxycytosine–doexyguanosine (CpG) motifs. GpG ODN are Toll-like receptor 9 (TLR9) ligands that stimulate antigen presenting dendritic cells (DC) [44]. Class A ODN stimulate high levels of INFα from plasmactyoid DC; B-class ODN induce strong B-cell and NK cell activation and the more recently discover C-class ODN appear have properties of both the A- and B-class ODN [45], [46]. Since several ODN adjuvants are in phase I human testing, we evaluated if B and C-class ODN, when formulated with protein in the boost phase of immunization, could augment the antibody response elicited by MF59 alone.

The guinea pig (GP) studies described here were designed to evaluate anti-Env antibody responses elicited by combinations of existing vaccine immunogens that have advanced into human clinical trails. Current HIV-1 DNA and rAd5 vectors are known to elicit robust T-cell responses in humans, but rather modest antibody levels. The soluble dV2gp140 protein elicits high antibody levels, but not CD8 T-cell responses. Thus, in human trials, it might be possible to establish a CD4 and CD8 T-cell response with the appropriate vaccine vectors, and further boost the antibody response with an Env protein. In these GP studies, we observed that a series of three DNA immunizations, or a single rAd5 immunization, produced moderate levels of anti-Env antibody, and effectively primed for boosting with soluble protein. A single protein boost immunization resulted in high antibody levels and the induction of virus neutralizing antibodies. The addition of a B- or C-class ODN to the protein formulation in the boost phase of immunization did not augment the antibody response generated by MF59 alone. While current Env based immunogens do not readily generate broadly reactive neutralizing antibodies, theses data suggest that DNA plus protein, or rAd5 plus protein, are valuable vaccine platforms that can be used to study the generation of HIV-1 anti-Env antibody responses. In addition, it may be possible to establish a T-cell response with appropriate vectored vaccines and further improve the neutralizing antibody titer with protein boosting.