Induction of HIV-1 subtype B and AE-specific neutralizing antibodies in mice and macaques with DNA prime and recombinant gp140 protein boost regimens
Introduction
Despite being the focus of much research effort, an effective vaccine to prevent HIV infection or ameliorate disease outcome after infection remains elusive. Env is the major viral target for neutralization by antibody. However Env has high sequence variability, extensive glycosylation and many of the more conserved Env structures are recessed, protecting the virus from neutralization. The first phase III clinical trial of an HIV vaccine used monomeric gp120 protein with the aim of generating neutralizing antibodies (nAb) to prevent infection. This vaccine was not protective [1], and it is now accepted that gp120 monomers induce only narrow nAb responses, primarily against CXCR4-tropic laboratory strains. Recently, efforts have focused on the potential of cytotoxic T lymphocyte (CTL) responses to control viremia and lower viral set point during chronic infection, thus preventing or delaying disease progression. Disappointingly, while a number of studies demonstrated the ability of vaccine-generated CTL responses to prevent or delay disease progression in non-human primate models, an adenoviral vector-based phase III clinical trial (STEP trial) recently failed to achieve these objectives [2]. Similarly, a previous approach from our group using a DNA prime and recombinant fowlpox virus boost failed to elicit significant adaptive cellular immune responses in human subjects [3]. In that study, a major truncation of Env (at amino acid 275) abrogated its ability to elicit anti-Env antibodies in macaques [4]. In the studies reported here we have tested immunogens with VLP-associated full-length Env and soluble Env analogues including the entire ectodomain (amino acids 1–683). VLP immunogens are recognized and taken up by antigen presenting cells, facilitating the generation of CD4 help and B-cell priming, whereas soluble immunogens can efficiently boost immune responses. VLP-producing plasmid DNA immunogens or purified VLP particles administered to macaques in the absence of subsequent heterologous boosting were able to mediate viral load reduction after homologous intravenous challenge, but not prevent infection [5], [6].
We have focused on generating broader humoral immune responses to Env. Env derived from both clade B and circulating recombinant form 01 AE (AE) strains were assessed in an attempt to increase the breadth of the immune responses elicited and tailor the vaccine for Thailand and other South East Asian countries where clade B and AE viruses are predominant. We tested our approach firstly in a murine model and then in a pilot study using pig-tailed macaques. Env was presented in DNA prime/trimeric protein boost regimens, as this has proven useful in eliciting strong and durable responses against Env and other immunogens, as reviewed by Lu [7]. DNA priming was designed to present Env in two different configurations: on the surface of virus like particles (VLP) and as a soluble Env analogue lacking the transmembrane and cytoplasmic domains (gp140). Our strategy elicited robust serum antibody responses that in the case of pig-tailed macaques showed some breadth of neutralization. However, immunization did not prevent infection after intrarectal challenge with a simian/human immunodeficiency virus expressing a heterologous HIV-1 Env (SHIVSF162P3), suggesting that eliciting broader immune responses in the mucosal compartment may be critical in future vaccine strategies.
Section snippets
DNA immunogens
The VLP-encoding plasmid pVLP was constructed by replacing the gag and pol genes of the plasmid pHIS-HIV-B [4] with those of pHIS-HIV-AE [8] using the restriction sites NotI and EcoRI, and restoring the truncated Env coding frame using an Nde1/AvrII fragment from the wild-type AD8 env gene [9] (Fig. 1A). To make plasmid vaccine vectors for the expression of soluble Env analogues, a BssHII/BlpI fragment of pDRenv2 [10] spanning the NL4.3 env gene was cloned into the same sites of pHIS-HIV-B,
In vitro expression from DNA vaccine vectors
A series of DNA and protein vaccines were designed to present native Env trimers and elicit nAb. The expression cassette of pVLP (Fig. 1A) retains approximately two-thirds of the proviral sequence. Multiple mutations or deletions of the gag, pol and nef coding regions and complete removal of the vpr and vif genes and both long terminal repeat sequences (all derived from the pHIS-HIV-B and pHIS-HIV-AE parental plasmids) ensured maximal safety. The expression cassettes of pVLP (Fig. 1A), pAD8-140
Discussion
In this study, we tested several modes of Env-based vaccine delivery in murine and non-human primate models. All plasmid vectors were tested in vitro by transfection of a human cell line, and high-level Env expression was confirmed. The VLP-encoding construct was very efficient at mediating secretion of VLP with high levels of incorporation of cleaved gp160, similar to that of virion-associated Env [20] indicating the potential utility of this vaccine platform. The murine system allowed us to
Acknowledgements
We thank Dr. Robert De Rose, Thakshila Amarasena, Caroline Fernandez, Jeanette Reece, Roberta Goli and Leanne Smith for expert technical assistance. This work was supported financially by the Australian NHMRC (Program Grant 299907 and Career Development Award 359233 to RJC) and the Australian Centre for HIV and Hepatitis Virology Research. The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: pSVIII-93.TH976.17 (for
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