A Chimeric HIV-1 gp120 Fused with Vaccinia Virus 14K (A27) Protein as an HIV Immunogen.

Aneesh Vijayan,Francisco Javier Chichón,Juan García-Arriaza,Mariano Esteban,José L Carrascosa,Carlos Óscar S Sorzano,José Javier Conesa,César Santiago,Suresh C Raman,Shan Lu

doi:10.1371/journal.pone.0133595

Aneesh Vijayan, Francisco Javier Chichón + Show 8 more

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https://doi.org/10.1371/journal.pone.0133595

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Abstract

In the HIV vaccine field, there is a need to produce highly immunogenic forms of the Env protein with the capacity to trigger broad B and T-cell responses. Here, we report the generation and characterization of a chimeric HIV-1 gp120 protein (termed gp120-14K) by fusing gp120 from clade B with the vaccinia virus (VACV) 14K oligomeric protein (derived from A27L gene). Stable CHO cell lines expressing HIV-1 gp120-14K protein were generated and the protein purified was characterized by size exclusion chromatography, electron microscopy and binding to anti-Env antibodies. These approaches indicate that gp120-14K protein is oligomeric and reacts with a wide spectrum of HIV-1 neutralizing antibodies. Furthermore, in human monocyte-derived dendritic cells (moDCs), gp120-14K protein upregulates the levels of several proinflammatory cytokines and chemokines associated with Th1 innate immune responses (IL-1β, IFN-γ, IL-6, IL-8, IL-12, RANTES). Moreover, we showed in a murine model, that a heterologous prime/boost immunization protocol consisting of a DNA prime with a plasmid expressing gp120-14K protein followed by a boost with MVA-B [a recombinant modified vaccinia virus Ankara (MVA) expressing HIV-1 gp120, Gag, Pol and Nef antigens from clade B], generates stronger, more polyfunctional, and greater effector memory HIV-1-specific CD4+ and CD8+ T-cell immune responses, than immunization with DNA-gp120/MVA-B. The DNA/MVA protocol was superior to immunization with the combination of protein/MVA and the latter was superior to a prime/boost of MVA/MVA or protein/protein. In addition, these immunization protocols enhanced antibody responses against gp120 of the class IgG2a and IgG3, together favoring a Th1 humoral immune response. These results demonstrate that fusing HIV-1 gp120 with VACV 14K forms an oligomeric protein which is highly antigenic as it activates a Th1 innate immune response in human moDCs, and in vaccinated mice triggers polyfunctional HIV-1-specific adaptive and memory T-cell immune responses, as well as humoral responses. This novel HIV-1 gp120-14K immunogen might be considered as an HIV vaccine candidate for broad T and B-cell immune responses.

Highlights

Acquired Immunodeficiency Syndrome (AIDS) is a scourge on mankind with an estimated 39 million deaths so far since the discovery of HIV-1, and over 35 million cases reported in 2013 (WHO Report October, 2014)
The purified HIV-1 gp120-14K proteins were run on SDS-PAGE under non-reducing conditions and the corresponding coomassie stained gels and western blots using antibodies against HIV-1 gp120 or vaccinia virus (VACV) 14K proteins showed that most gp120-14K proteins appear as high molecular weight (Fig 1B), with subtle differences observed in the size of monomers derived from Chinese Hamster Ovary (CHO)-K1 versus CHO-Lec cells, probably due to different glycosylation or mannose content
The results showed that genes influencing Th1 innate immune responses, such as IL-1β, IL-8, RANTES, IFN-γ, IL6 and IL-12p40 were significantly upregulated by gp120-14KCHO-K1 at 3 and 6 h post-treatment, in comparison to mock cells or to monomeric gp120 protein (Fig 4A)

Summary

Introduction

Acquired Immunodeficiency Syndrome (AIDS) is a scourge on mankind with an estimated 39 million deaths so far since the discovery of HIV-1, and over 35 million cases reported in 2013 (WHO Report October, 2014). Among the different HIV/AIDS vaccine candidates developed, the HIV-1 envelope glycoprotein stands out to be the most promising one [3, 4]. Generating an Env protein which mimics the native conformation is a long sought goal in HIV/AIDS vaccine development since the use of monomeric gp120 in clinical trials ended in failures with the exception of RV144 phase III clinical trial that showed a modest efficacy of 31.2% [11]. The conformational differences between the purified monomeric gp120 protein and its native form could explain these failures. Some of the recently identified bnAbs, which bind exclusively to gp120 trimer, hold the key for rapid screening of potent vaccine candidates [13, 15]. PG9 and PG16, glycan dependent immunoglobulins isolated from an African donor, recognize an epitope on the quaternary structure of the gp120 protein [15, 16]

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