Elsevier

Vaccine

Volume 27, Issue 6, 5 February 2009, Pages 857-863
Vaccine

Cross-reactive HIV-1-neutralizing activity of serum IgG from a rabbit immunized with gp41 fused to IgG1 Fc: Possible role of the prolonged half-life of the immunogen

https://doi.org/10.1016/j.vaccine.2008.11.083Get rights and content

Abstract

The elicitation of broadly cross-reactive HIV-1 neutralizing antibodies in humans remains a major challenge in developing a viable AIDS vaccine. We hypothesized that prolonged exposure to candidate vaccine immunogens could enhance the elicitation of such antibodies. In an attempt to develop HIV-1 vaccine immunogens with prolonged half-lives and increased stability, we constructed a fusion protein, gp41Fc, in which a truncated HIV-1 gp4189.6 was fused to a human IgG1 Fc. Gp41Fc is stable in solution, retains its antigenic structure and is highly immunogenic in rabbits. The serum titers reached 1:102,400 for the gp41Fc and 1:5,120 for gp14089.6. Rabbit IgG neutralized diverse HIV-1 isolates and HIV-2, and the neutralization activity was attributed to gp41-specific IgG. The concentration of the gp41Fc in the serum correlated with the neutralization activity of rabbit IgG which recognized mostly conformation-independent epitopes on gp41 and predominantly bound to peptides derived from the gp41 immunodominant loop region. These results suggest that the prolonged half-life of gp41Fc in the serum may enhance the generation of cross-reactive neutralizing antibodies. Further research is needed to confirm and extend these results which may have implications for the development of vaccine immunogens with enhanced capability to elicit cross-reactive HIV-1-neutralizing antibodies.

Introduction

Human immunodeficiency virus type 1 (HIV-1) has become a growing pandemic since its discovery in the early 1980s. After more than two decades of an intensive search for a viable AIDS vaccine, the goal remains elusive. The elicitation of broadly cross-reactive HIV-1 neutralizing antibodies in humans remains a major challenge in this effort largely due to HIV-1 genetic diversity.

The HIV-1 envelope glycoprotein (Env) is known to exist as trimers of heterodimer on native virions that is composed of a non-covalently associated extracellular subunit gp120 and a transmembrane subunit gp41. In natural infections, Env induces a robust antibody response, but these antibodies usually have a narrow spectrum of neutralization activity. Occasionally some HIV-1-positive individuals induce broadly cross-reactive neutralizing antibodies that are believed to account for the containment of the virus [1], [2]. Env-based vaccines have been extensively studied. Monomeric gp120 s can elicit neutralizing antibodies in vivo, but the neutralization activity is restricted to autologous viruses or the spectrum of neutralization activity is narrow [3]. Oligomeric forms of Env ectodomain from HIV-1, strain R2 (gp140R2), which contain both gp120 and a truncated gp41 that lacks both transmembrane domains and cytoplasmic tails, induced better antibody response than gp120R2 alone. Rabbit sera immunized with gp140R2 neutralized not only the autologous virus, but also heterologous viruses from diverse HIV-1 subtypes, suggesting that induction of broad spectrum neutralizing antibodies is an achievable goal in HIV-1 vaccine development [4].

This study utilizes gp41 derived from a dual tropic HIV-1 primary isolate of 89.6. Gp41 is relatively conserved compared to gp120, but unstable in the absence of gp120. To stabilize gp41 in the absence of gp120, we constructed a fusion protein, designated as gp41Fc, in which a truncated gp4189.6 that lacked a fusion peptide, transmembrane domain and cytoplasmic tail was fused to human Fc by a long flexible linker. The goal of this fusion protein was to increase its half-life in vivo and enhance its binding to immune cells like macrophages, dendritic cells, and B cells that express receptors specific for Fc [5]. We hypothesize that prolonged exposure to candidate vaccine immunogens could enhance the elicitation of broadly cross-reactive antibodies based on the observation that broadly cross-reactive HIV-1 neutralizing antibodies are typically elicited later than isolate-specific antibodies after infection. Initially gp41Fc was used to localize the epitopes of a panel of gp41-specific neutralizing antibodies by alanine scanning mutagenesis. We found that gp41Fc not only bound to our recently identified monoclonal antibodies (mAbs) recognizing conformational epitopes, m44 [6], m46 [7] and m48 [8], but also to known broadly neutralizing human mAbs that include 2F5, 4E10, Z13 that recognize linear epitopes in the membrane proximal region (MPER), suggesting that gp41Fc retains critical conformation required for presenting neutralization epitopes and may induce neutralizing antibodies against HIV-1 in vivo. This prompted us to test the possibility of developing this fusion protein as HIV-1 vaccine immunogen by immunizing rabbits with purified recombinant gp41Fc. Here we report the results from the characterization of immune rabbit sera and purified IgGs from two rabbits that had different immune response to gp41Fc.

Section snippets

Cells, viruses, plasmids, gp120, gp140 and antibodies

293T cells were purchased from ATCC. Other cell lines and HIV-1 isolates were obtained from the NIH AIDS Research and Reference Reagent Program (ARRRP). Recombinant gp120 and gp140 Envs from primary isolates were produced as described previously [9]. Human mAbs 2F5 and 4E10 were obtained from the ARRRP. Mouse mAbs NC-1 was generously provided by Dr. Shibo Jiang (New York Blood Center). Mouse mAbs T3 and D61 were obtained from Dr. Christopher Broder (Uniformed Services University of the Health

Recombinant gp41Fc retains its antigenic structure

Purified gp41Fc separated on SDS-PAGE gels appear as a single band on denatured gels and several higher order bands on non-denatured gels. These results indicate the existence of oligomers, which could contribute to its avidity (data not shown).

Recombinant gp41Fc retains its antigenic structure as measured by ELISA (Fig. 1). Recombinant gp41Fc binds to all gp41-specific mAbs tested including human mAbs m44, m46 and m48, and mouse NC-1 and T3 that recognize the conformational epitopes of gp41,

Discussion

This study describes the results from two rabbits immunized with a gp41Fc fusion protein. The major observation of this study is that prolonged existence of the immunogen in rabbit serum correlates with the neutralization activity of rabbit IgG. Although data from more animals is required to derive statistically significant data, these results suggest that prolonging the existence of the immunogen in vivo may enhance elicitation of broadly cross-reactive HIV-1 neutralizing Abs. This hypothesis

Acknowledgements

We thank Dr. Shibo Jiang for providing mouse mAb NC-1 and Dr. Christopher Broder for mouse mAbs T3 and D61. We also thank Drs. Shibo Jiang and Xiaodong Xiao for helpful discussions, Vidita Choudhry for help with pseudovirus assay and John Owens for help with manuscript preparation. We thank one of the reviewers for suggesting to explore gp140Fc fusion proteins as vaccine immunogens. This research was supported by the NIH Intramural AIDS Targeted Antiviral Program (IATAP), the Intramural

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Center for Cancer Research Nanobiology Program, CCR, NCI-Frederick, NIH, Bldg 469, Rm 150B, P.O. Box B, Miller Drive, Frederick, MD 21702-1201, United States. Tel.: +301 846 1352; fax: +301 846 5598.

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