Engineering Recombinant Reoviruses To Display gp41 Membrane-Proximal External-Region Epitopes from HIV-1

Ikizler, Mine R.; Iskarpatyoti, Jason A.; Wetzel, J. Denise; Willis, Jordan R.; Crowe, James E.; LaBranche, Celia C.; Montefiori, David C.; Wilson, Gregory J.; Dermody, Terence S.

doi:10.1128/msphere.00086-16

Cited by 5 publications

(2 citation statements)

References 95 publications

(123 reference statements)

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“…We and others have found that genetic modification of reovirus can improve oncolytic activity in vitro and in animal cancer models (87)(88)(89)(90)(91). Reovirus also provides a potential vaccine vector (92,93). Efficient production of reovirus recombinants will therefore expedite production and testing of genetically modified reovirus for oncolytic and vaccination purposes.…”

Section: Discussionmentioning

confidence: 99%

African Swine Fever Virus NP868R Capping Enzyme Promotes Reovirus Rescue during Reverse Genetics by Promoting Reovirus Protein Expression, Virion Assembly, and RNA Incorporation into Infectious Virions

Eaton

Kobayashi

Dermody

et al. 2017

J Virol

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Reoviruses, like many eukaryotic viruses, contain an inverted 7-methylguanosine (m7G) cap linked to the 5' nucleotide of mRNA. The traditional functions of capping are to promote mRNA stability, protein translation, and concealment from cellular proteins that recognize foreign RNA. To address the role of mRNA capping during reovirus replication, we assessed the benefits of adding the African swine fever virus NP868R capping enzyme during reovirus rescue. C3P3, a fusion protein containing T7 RNA polymerase and NP868R, was found to increase protein expression 5- to 10-fold compared to T7 RNA polymerase alone while enhancing reovirus rescue from the current reverse genetics system by 100-fold. Surprisingly, RNA stability was not increased by C3P3, suggesting a direct effect on protein translation. A time course analysis revealed that C3P3 increased protein synthesis within the first 2 days of a reverse genetics transfection. This analysis also revealed that C3P3 enhanced processing of outer capsid μ1 protein to μ1C, a previously described hallmark of reovirus assembly. Finally, to determine the rate of infectious-RNA incorporation into new virions, we developed a new recombinant reovirus S1 gene that expressed the fluorescent protein UnaG. Following transfection of cells with UnaG and infection with wild-type virus, passage of UnaG through progeny was significantly enhanced by C3P3. These data suggest that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation. Our findings expand our understanding of how viruses utilize capping, suggesting that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation, in addition to enhancing protein translation. Beyond providing mechanistic insight into reovirus replication, our findings also show that reovirus reverse genetics rescue is enhanced 100-fold by the NP868R capping enzyme. Since reovirus shows promise as a cancer therapy, efficient reovirus reverse genetics rescue will accelerate production of recombinant reoviruses as candidates to enhance therapeutic potency. NP868R-assisted reovirus rescue will also expedite production of recombinant reovirus for mechanistic insights into reovirus protein function and structure.

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Section: Discussionmentioning

confidence: 99%

African Swine Fever Virus NP868R Capping Enzyme Promotes Reovirus Rescue during Reverse Genetics by Promoting Reovirus Protein Expression, Virion Assembly, and RNA Incorporation into Infectious Virions

Eaton

Kobayashi

Dermody

et al. 2017

J Virol

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“…The MPER is a highly conserved region targeted by broadly neutralizing antibodies (bnAbs) [ 100 ]. Although such MPER-specific antibodies have been shown to prevent infection through passive immunization [ 101 , 102 , 103 ], numerous animal studies have failed to elicit robust or sufficiently broad neutralizing antibody responses using a variety of strategies [ 104 , 105 , 106 , 107 , 108 , 109 , 110 , 111 , 112 , 113 , 114 ]. Issues hindering the development of gp41 MPER as a vaccine target include poor immunogenicity due in part to steric hindrance and lack of accessibility [ 100 , 115 ], hydrophobicity that renders the MPER prone to aggregation in solution [ 116 ], immunodominance of adjacent gp41 regions lacking any neutralizing epitopes [ 117 , 118 ] and the apparent auto-reactivity of MPER-specific bnAbs towards cell membrane lipids [ 119 , 120 ].…”

Section: Introductionmentioning

confidence: 99%

DNA Vaccine-Encoded Flagellin Can Be Used as an Adjuvant Scaffold to Augment HIV-1 gp41 Membrane Proximal External Region Immunogenicity

Ajamian

Melnychuk

Jean-Pierre

et al. 2018

Viruses

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Flagellin’s potential as a vaccine adjuvant has been increasingly explored over the last three decades. Monomeric flagellin proteins are the only known agonists of Toll-like receptor 5 (TLR5). This interaction evokes a pro-inflammatory state that impacts upon both innate and adaptive immunity. While pathogen associated molecular patterns (PAMPs) like flagellin have been used as stand-alone adjuvants that are co-delivered with antigen, some investigators have demonstrated a distinct advantage to incorporating antigen epitopes within the structure of flagellin itself. This approach has been particularly effective in enhancing humoral immune responses. We sought to use flagellin as both scaffold and adjuvant for HIV gp41 with the aim of eliciting antibodies to the membrane proximal external region (MPER). Accordingly, we devised a straightforward step-wise approach to select flagellin-antigen fusion proteins for gene-based vaccine development. Using plasmid DNA vector-based expression in mammalian cells, we demonstrate robust expression of codon-optimized full length and hypervariable region-deleted constructs of Salmonella enterica subsp. enterica serovar Typhi flagellin (FliC). An HIV gp41 derived sequence including the MPER (gp41607–683) was incorporated into various positions of these constructs and the expressed fusion proteins were screened for effective secretion, TLR5 agonist activity and adequate MPER antigenicity. We show that incorporation of gp41607–683 into a FliC-based scaffold significantly augments gp41607–683 immunogenicity in a TLR5 dependent manner and elicits modest MPER-specific humoral responses in a mouse model.

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The development of HIV vaccines targeting gp41 membrane-proximal external region (MPER): challenges and prospects

Liu

et al. 2018

Protein &Amp; Cell

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A human immunodeficiency virus type-1 (HIV-1) vaccine which is able to effectively prevent infection would be the most powerful method of extinguishing pandemic of the acquired immunodeficiency syndrome (AIDS). Yet, achieving such vaccine remains great challenges. The membrane-proximal external region (MPER) is a highly conserved region of the envelope glycoprotein (Env) gp41 subunit near the viral envelope surface, and it plays a key role in membrane fusion. It is also the target of some reported broadly neutralizing antibodies (bNAbs). Thus, MPER is deemed to be one of the most attractive vaccine targets. However, no one can induce these bNAbs by immunization with immunogens containing the MPER sequence(s). The few attempts at developing a vaccine have only resulted in the induction of neutralizing antibodies with quite low potency and limited breadth. Thus far, vaccine failure can be attributed to various characteristics of MPER, such as those involving structure and immunology; therefore, we will focus on these and review the recent progress in the field from the following perspectives: (1) MPER structure and its role in membrane fusion, (2) the epitopes and neutralization mechanisms of MPER-specific bNAbs, as well as the limitations in eliciting neutralizing antibodies, and (3) different strategies for MPER vaccine design and current harvests.

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Engineering Recombinant Reoviruses To Display gp41 Membrane-Proximal External-Region Epitopes from HIV-1

Cited by 5 publications

References 95 publications

African Swine Fever Virus NP868R Capping Enzyme Promotes Reovirus Rescue during Reverse Genetics by Promoting Reovirus Protein Expression, Virion Assembly, and RNA Incorporation into Infectious Virions

African Swine Fever Virus NP868R Capping Enzyme Promotes Reovirus Rescue during Reverse Genetics by Promoting Reovirus Protein Expression, Virion Assembly, and RNA Incorporation into Infectious Virions

DNA Vaccine-Encoded Flagellin Can Be Used as an Adjuvant Scaffold to Augment HIV-1 gp41 Membrane Proximal External Region Immunogenicity

The development of HIV vaccines targeting gp41 membrane-proximal external region (MPER): challenges and prospects

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