Bovine papillomavirus-like particles presenting conserved epitopes from membrane-proximal external region of HIV-1 gp41 induced mucosal and systemic antibodies
Introduction
Although anti-HIV-1 (human immunodeficiency virus type 1) drugs are effective in controlling development of AIDS, HIV-1 infection still remains an enormous problem worldwide. An effective HIV-1 vaccine is urgently needed but its development is hindered by the huge variability of the virus. A recent HIV-1 vaccine phase III clinical trial, the RV144 trial conducted in Thailand, for the first time showed modest efficacy against acquisition of infection, which raises hope for developing an effective HIV-1 vaccine [1], [2].
A number of groups have isolated several monoclonal antibodies (mAbs) that can neutralize a variety of HIV-1 isolates [3], [4], [5], [6], [7], [8], [9], [10], [11], [12]. Among those mAbs, 2F5, 4E10, Z13 and the recently reported 10E8 are shown to target conserved epitopes within the membrane-proximal external region (MPER) of gp41 [12], [13], [14], [15], [16], [17], [18], [19], [20], [21]; The isolation of these and other broadly neutralizing antibodies (b12, 2G12, VRC01, CH01 to CH04, PG9, PG16) is a milestone in the field. The epitopes located at the MPER of gp41 recognized by broadly neutralizing mAbs 2F5 and 4E10 are promising targets for vaccine design. Unfortunately, many attempts at eliciting broadly neutralizing antibodies by targeting neutralizing epitopes of MPER failed [22], [23], [24], [25], [26], [27]. Two recent studies reported that presenting the ELDKWA epitope by human rhinovirus [28] or a whole MPER domain with mutations (T569A and I675V) by NCM antigen [29] successfully induced anti-HIV-1 cross-clade neutralizing antibodies.
HIV-1 is transmitted both venereally and hematogeneously and vaginal and rectal mucosa serve as major sites for viral entry and initial infection [30], [31]. Thus, an effective HIV-1 vaccine must elicit both mucosal immunity to prevent entry of HIV-1 into the mucosa, and systemic immunity to restrict viral transmission via blood stream [32]. Bovine papillomavirus 1 (BPV) virus-like particle (VLP) is an ideal mucosal delivery vector that not only carries peptide/protein/DNA to protect them from degradation by the gastric and intestinal environment but also serves as a strong immune activating adjuvant eliciting both mucosal and systemic immune responses via the oral immunization route [27], [33], [34], [35], [36], [37]. Additionally, since BPV is not a natural pathogen for humans, the likelihood of preexisting “vector neutralization” that would presumably attenuate vaccine efficacy is minimized.
In this study, we used BPV VLPs to present HIV-1 gp41 MPER or extended 2F5 epitope and determined if the epitopes are presented in their proper conformation. Induction of systemic and mucosal antibody responses was measured after oral immunization of the chimerical VLPs in mice. We used HIV-1 infected cells and an in vitro neutralization test to assess the binding and neutralizing activities of the induced HIV-1-specific antibodies.
Section snippets
Cell lines
Sf9 (Spodoptera frugiperda) cells were obtained from the American Type Culture Collection and grown at 28 °C in HyGro Sf-PFM insect medium (JR Scientific, Woodland, CA). Human T-lymphoblastic cell lines H9 and CEM-NKR-CCR5 were obtained from the AIDS Research and Reference Reagent Program, NIH [38], [39]. T cells were cultured in L-glutamine containing DMEM medium supplemented with 10% fetal bovine serum, 100 U of penicillin and 100 μg of streptomycin per ml (HyClone, Logan, UT).
Construction and generation of CVLPs
The C-terminal
Design and generation of CVLPs
We used BPV1 L1 VLPs to present HIV gp41 neutralizing epitopes to develop an HIV-1 vaccine. We used an extended 13aa 2F5 epitope, 13aa 4E10 epitope or an MPER peptide containing both epitopes in the vaccine constructs. Three chimeric L1 VLPs were made: 2-1 (presenting ELLELDKWASLWN), 4-1 (presenting NWFDITNWLWYIK) and L1-MPER (presenting ELLELDKWASLWNWFDITNWLWYIK) (Fig. 1A). Purified CVLPs were identified by western-blotting using anti-L1 mAb AU5 (Fig. 1B). Western-blot using 2F5 or 4E10 mAb
Discussion
In this study, we developed chimeric BPV1-gp41 to elicit mucosal and systemic anti-HIV-1 broadly neutralizing antibodies. We inserted the extended 2F5 and 4E10 epitopes as well as MPER domain containing three neutralizing epitopes, into the D-E loop of BPV L1 to broaden the presentation of the HIV-1 2F5/Z13/4E10 epitope on the BPV CVLPs as well as the protection against HIV-1. Our results demonstrated that the MPER-displaying CVLPs induced HIV-specific mucosal and systemic cross-clade
Acknowledgments
The following reagents were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 gp41 mAbs 2F5 and 4E10 from Dr. Hermann Katinger; H9 cells, HIV-1MN and HIV-1Bal from Dr. Robert Gallo; HIV-1SE364, UG273, UG274, US4 from Dr. Nelson Michael. This work was supported by a grant from the National Institutes of Health (NIH) (grant # DE019075 to L.Q.).
Conflict of interest: The authors have declared that no financial conflict of interest exists.
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