All-in-One Bacmids: an Efficient Reverse Genetics Strategy for Influenza A Virus Vaccines

Chen, Hongjun; Angel, Matthew; Li, Weizhong; Finch, Courtney; González, Ana Silvia; Sutton, Troy; Santos, Jefferson; Pérez, Daniel R.

doi:10.1128/jvi.01468-14

Cited by 20 publications

(20 citation statements)

References 62 publications

(63 reference statements)

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“…In vivo rescue of rZiKV by inoculation of cells transfected with pBAc-ZiKV. Chen et al had previously described a method for the in vivo recovery of influenza A virus (IAV), a negative-strand RNA virus, after the inoculation in the nasal cavity of mice with cells transfected with a bacmid containing the entire IAV genome 34 . A similar approach was used to study the potential for reassortment between different IAV subtype strains in ferrets 35,36 .…”

Section: Resultsmentioning

confidence: 99%

In vivo rescue of recombinant Zika virus from an infectious cDNA clone and its implications in vaccine development

Ávila‐Pérez

Nogales

Park

et al. 2020

Sci Rep

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Zika virus (ZiKV) is a mosquito-borne member of theFlaviviridae family that has been known to circulate for decades causing mild febrile illness. the more recent ZiKV outbreaks in the Americas and the caribbean associated with congenital malformations and Guillain-Barré syndrome in adults have placed public health officials in high alert and highlight the significant impact of ZIKV on human health. New technologies to study the biology of ZIKV and to develop more effective prevention options are highly desired. in this study we demonstrate that direct delivery in mice of an infectious ZiKV cDnA clone allows the rescue of recombinant (r)ZiKV in vivo. A bacterial artificial chromosome containing the sequence of ZIKV strain Paraiba/2015 under the control of the cytomegalovirus promoter was complexed with a commercial transfection reagent and administrated using different routes in type-I interferon receptor deficient A129 mice. Clinical signs and death associated with ZIKV viremia were observed in mice. the rZiKV recovered from these mice remained fully virulent in a second passage in mice. interestingly, infectious rZiKV was also recovered after intraperitoneal inoculation of the rZiKV cDnA in the absence of transfection reagent. further expanding these studies, we demonstrate that a single intraperitoneal inoculation of a cDnA clone encoding an attenuated rZiKV was safe, highly immunogenic, and provided full protection against lethal ZiKV challenge. this novel in vivo reverse genetics method is a potentially suitable delivery platform for the study of wild-type and liveattenuated ZiKV devoid of confounding factors typical associated with in vitro systems. Moreover, our results open the possibility of employing similar in vivo reverse genetic approaches for the generation of other viruses and, therefore, change the way we will use reverse genetics in the future.Zika virus (ZIKV), a member of the Flaviviridae family, became a global public concern because of the correlation of Zika virus epidemic with fetal developmental defects, including highly publicized cases of microcephaly 1 . The viral genome is made of a positive sense, single-stranded RNA molecule (~10.8 kb) that contains a single open reading frame flanked by 5′ and 3′ untranslated regions 2-4 . The viral RNA is translated as a single polyprotein that is co-and post-translationally processed by viral and cellular proteases into three major structural proteins (capsid, pre-membrane and envelope) involved in viral entry, fusion and assembly 5 , and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) that are involved in viral RNA replication and transcription, assembly and evasion of the host antiviral responses [6][7][8][9] .Although the majority of ZIKV infections are asymptomatic or associated with a mild febrile illness, infection during pregnancy has been associated with miscarriage and severe congenital malformations, including fetal

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Section: Resultsmentioning

confidence: 99%

In vivo rescue of recombinant Zika virus from an infectious cDNA clone and its implications in vaccine development

Ávila‐Pérez

Nogales

Park

et al. 2020

Sci Rep

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“…Most recently, we constructed all-in-one bacmids driven by hpol1 using Gateway cloning technology, which were used to overcome some of the limitations of cloning full influenza virus RG sets into a single DNA unit constrained by stability or copy number. bcmd-RGFlu constructs succeeded in improving transfection efficiency (100-fold) compared to the eight-plasmid counterparts, particularly in hard-to-transfect Vero cells [17]. In this report, we initially tried to develop a modified RG system based on the spol1 promoter, which was supposed to have higher efficiency.…”

Section: Discussionmentioning

confidence: 99%

“…The recombinant cloning strategy of the RG eightplasmid system for the HN07 virus has been described previously [17]. The segments were amplified with the primers shown in Table 1, and subcloned into pHC2014 using a rapid restriction enzyme-free Exnase TM II-based in vitro recombination approach from our previous study [21].…”

Section: Subcloning Using An In Vitro Recombination Approachmentioning

confidence: 99%

“…Some modifications have been used to improve vaccine generation in mammalian cells, one of which is to construct degenerated plasmids [15,16]. Our previous study successfully improved the efficiency of using Gateway cloning strategy to subclone all essential elements from an eightplasmid RG system using several candidates as donor vectors [17]. Another way to improve RG efficiency in licensed cell lines is to replace the RNA pol1, for which the polymerase from mice [18], chickens [16], horses [19], dogs [13], and monkeys [14] have been used to construct RG systems.…”

Section: Introductionmentioning

confidence: 99%

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Alternative reverse genetics system for influenza viruses based on a synthesized swine 45S rRNA promoter

et al. 2017

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We generated an alternative reverse genetics (RG) system based on a synthesized swine 45S rRNA promoter to rescue the H3N2 subtype swine influenza virus. All eight flanking segment cassettes of A/swine/ Henan/7/2010 (H3N2) were amplified with ambisense expression elements from RG plasmids. All segments were then recombined with the pHC2014 vector, which contained the synthesized swine 45S rRNA promoter (spol1) and its terminal sequence (t1) in a pcDNA3 backbone. As a result, we obtained a set of RG plasmids carrying the corresponding eight-segment cassettes. We efficiently generated the H3N2 virus after transfection into 293T/ PK15, PK15, and 293T cells. The efficiency of spol1-driven influenza virus rescue in PK15 cells was similar to that in 293T cells by titration using the human pol1 RG system. Our approach suggests that an alternative spol1-based RG system can produce influenza viruses.

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“…The influenza virus surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) are key mediators of the viral infectious cycle and obvious proteins of interest for novel vaccines. The majority of recent vaccine candidates focus predominantly on the immunogenic properties of the influenza virus HA, and sometimes NA, glycoprotein, seeking to utilize vector systems (1)(2)(3), adjuvants (4)(5)(6), alternative routes of immunization (7,8), and novel antigen presentation and delivery systems (9)(10)(11)(12) to increase humoral and/or cell-mediated immunity against these and other influenza virus antigens. Creating vaccines that also exploit the functional properties of the influenza virus glycoproteins represents a unique approach.…”

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confidence: 99%

A Viable Recombinant Rhabdovirus Lacking Its Glycoprotein Gene and Expressing Influenza Virus Hemagglutinin and Neuraminidase Is a Potent Influenza Vaccine

Ryder

Buonocore

Vogel

et al. 2015

J Virol

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The emergence of novel influenza viruses that cause devastating human disease is an ongoing threat and serves as an impetus for the continued development of novel approaches to influenza vaccines. Influenza vaccine development has traditionally focused on producing humoral and/or cell-mediated immunity, often against the viral surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Here, we describe a new vaccine candidate that utilizes a replication-defective vesicular stomatitis virus (VSV) vector backbone that lacks the native G surface glycoprotein gene (VSV⌬G). The expression of the H5 HA of an H5N1 highly pathogenic avian influenza virus (HPAIV), A/Vietnam/1203/04 (VN1203), and the NA of the mouse-adapted H1N1 influenza virus A/Puerto Rico/8/34 (PR8) in the VSV⌬G vector restored the ability of the recombinant virus to replicate in cell culture, without the requirement for the addition of trypsin. We show here that this recombinant virus vaccine candidate was nonpathogenic in mice when given by either the intramuscular or intranasal route of immunization and that the in vivo replication of VSV⌬G-H5N1 is profoundly attenuated. This recombinant virus also provided protection against lethal H5N1 infection after a single dose. This novel approach to vaccination against HPAIVs may be widely applicable to other emerging strains of influenza virus. IMPORTANCEPreparation for a potentially catastrophic influenza pandemic requires novel influenza vaccines that are safe, can be produced and administered quickly, and are effective, both soon after administration and for a long duration. We have created a new influenza vaccine that utilizes an attenuated vesicular stomatitis virus (VSV) vector, to deliver and express influenza virus proteins against which vaccinated animals develop potent antibody responses. The influenza virus hemagglutinin and neuraminidase proteins, expressed on the surface of VSV particles, allowed this vaccine to grow in cell culture and induced a potent antibody response in mice that was effective against infection with a lethal influenza virus. The mice showed no adverse reactions to the vaccine, and they were protected against an otherwise lethal influenza infection after only 14 days postvaccination and after as many as 140 days postvaccination. The ability to rapidly produce this safe and effective vaccine in cell culture is additionally advantageous.

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All-in-One Bacmids: an Efficient Reverse Genetics Strategy for Influenza A Virus Vaccines

Cited by 20 publications

References 62 publications

In vivo rescue of recombinant Zika virus from an infectious cDNA clone and its implications in vaccine development

In vivo rescue of recombinant Zika virus from an infectious cDNA clone and its implications in vaccine development

Alternative reverse genetics system for influenza viruses based on a synthesized swine 45S rRNA promoter

A Viable Recombinant Rhabdovirus Lacking Its Glycoprotein Gene and Expressing Influenza Virus Hemagglutinin and Neuraminidase Is a Potent Influenza Vaccine

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