Seamless manipulation of the varicella-zoster virus genome via bacterial artificial chromosome engineering

The human alphaherpesvirus varicella-zoster virus (VZV) causes chickenpox during primary infection and shingles upon reactivation from its latent state in sensory ganglia. Shingles are often associated with severe pain that can persist as postherpetic neuralgia. In order to facilitate the investigation of VZV, we constructed the genomes of the VZV strains P-Oka and HJO as infectious F-factor-derived bacterial artificial chromosomes (BACs). These two VZV BACs (pP-Oka and pHJO) can be seamlessly manipulated by versatile recombination techniques in Escherichia coli and subsequently delivered into VZV-permissive eukaryotic cells to allow the reconstitution of infectious mutant virus. In this work, the two different VZV BACs were engineered in E. coli to induce the seamless removal of the bacterial mini-F sequences upon virus reconstitution in VZV-permissive cells via novel replication-based strategies. In pP-Oka-derived virus, the mini-F sequences were efficiently released by homologous recombination of a stabilized inverse genomic duplication inserted into the bacterial vector elements. In contrast, this system was ineffective in virus progeny recovered from pHJO. To increase the efficiency of the vector elimination upon virus reconstitution from the cloned HJO genome, the mini-F sequences were precisely and seamlessly transposed within the viral DNA via a novel synchronous Red-recombination reaction in E. coli, whereby six different BAC variants were generated. The insertion of the mini-F sequences directly between the genomic termini resulted in a BAC derivate, of which the vector elements were autonomously and efficiently released during the virus recon¬stitution even without a genomic duplication. In addition, the derived progeny exhibited genome properties and replication kinetics virtually identical to that of the wild-type virus. In conclusion, the pP-Oka BAC with an inverse genomic duplication and the BAC variant of pHJO with terminal integrated mini-F vector are two optimized constructs to rapidly generate VZV mutants without leaving behind any operational sequences. Moreover, the mini-F vector transposition reaction eliminates the last hurdle to perform virtually any kind of imaginable targeted and seamless BAC modifications in E. coli. Thus, the newly developed manipulation techniques can be useful to optimize, repair, or restructure other established BACs as well, not even for the specific research field of herpesviruses, which may facilitate the development of gene therapy and vaccine vectors.Das menschliche Alphaherpesvirus Varicella-Zoster-Virus (VZV) verursacht Windpocken bei der Primärinfektion und Gürtelrose bei der Reaktivierung aus der Latenz in sensorischen Ganglien. Die Gürtelrose ist häufig mit schweren Schmerzen verbunden, die als postherpeti-sche Neuralgie persistieren können. Um die Erforschung von VZV zu erleichtern, klonierten wir die Genome der VZV-Stämme P-Oka und HJO als infektiöse, F-Plasmid-abgeleitete bakterielle artifizielle Chromosomen (BACs). Diese beiden VZV-BACs (pP-Oka und pHJO) können durch vielseitig anwendbare Rekombinationstechniken in Escherichia coli nahtlos manipuliert und anschließend in VZV-permissive eukaryote Zellen transfiziert werden, um die Rekonstitution von infektiösen Virusmutanten zu ermöglichen. In dieser Arbeit wurden die beiden unterschiedlichen VZV-BACs in E. coli verändert, um die nahtlose Entfernung der bakteriellen Mini-F-Sequenzen nach der Virusrekonstitution durch neuartige Replikationsstrategien zu induzieren. Bei aus pP-Oka hergestelltem Virus wurden die Mini-F-Elemente durch homologe Rekombination einer inversen stabilisierten geno-mischen Duplikation im bakteriellen Vektoranteil effizient entfernt. Im Gegensatz dazu war dieses System in Viren hergestellt aus pHJO ineffektive. Um die Effizienz der Vektor-elimination nach der Virusrekonstitution aus dem klonierten HJO-Genom zu verstärken, wurden die Mini-F-Sequenzen durch eine neuartige, synchrone Red-Rekombinationsreaktion in E. coli innerhalb der viralen DNA transponiert, wodurch sechs verschiedene BAC-Varianten konstruiert wurden. Die Insertion der Mini-F-Sequenzen direkt zwischen die Genomenden resultierte in einer BAC-Variante, von der die Vektorsequenzen autonom und effizient nach der Virusrekonstitution entfernt wurden, auch ohne genomische Duplikation. Zudem zeigten die hergestellten Viren Genomeigenschaften und Replikationskinetiken, die mit denen vom Wildtyp-Virus identisch waren. Der pP-Oka BAC mit inverser genomischer Duplikation und die BAC-Variante von pHJO mit terminal integriertem Mini-F-Vektor sind somit zwei optimierte Konstrukte zur schnellen Herstellung von Virusmutanten, ohne irgendwelche Fremdsequenzen zu hinterlassen. Zudem eliminiert die Mini-F-Transpositionsreaktion die letzte Hürde um jede vorstellbare, nahtlose BAC-Modifikation in E. coli durchzuführen. Die neuentwickelten Manipulationstechniken können somit nützlich sein um auch andere etablierte BACs zu optimieren, reparieren oder restrukturieren, nicht nur für das spezielle Forschungsgebiet der Herpesviren, was die Ent-wicklung von Gentherapie- oder Impfstoffvektoren erleichtern könnte

Red-Mediated Transposition and Final Release of the Mini-F Vector of a Cloned Infectious Herpesvirus Genome

Bacterial artificial chromosomes (BACs) are well-established cloning vehicles for functional genomics and for constructing targeting vectors and infectious viral DNA clones. Red-recombination-based mutagenesis techniques have enabled the manipulation of BACs in Escherichia coli without any remaining operational sequences. Here, we describe that the F-factor-derived vector sequences can be inserted into a novel position and seamlessly removed from the present location of the BAC-cloned DNA via synchronous Red-recombination in E. coli in an en passant mutagenesis-based procedure. Using this technique, the mini-F elements of a cloned infectious varicella zoster virus (VZV) genome were specifically transposed into novel positions distributed over the viral DNA to generate six different BAC variants. In comparison to the other constructs, a BAC variant with mini-F sequences directly inserted into the junction of the genomic termini resulted in highly efficient viral DNA replication-mediated spontaneous vector excision upon virus reconstitution in transfected VZV-permissive eukaryotic cells. Moreover, the derived vector-free recombinant progeny exhibited virtually indistinguishable genome properties and replication kinetics to the wild-type virus. Thus, a sequence-independent, efficient, and easy-to-apply mini-F vector transposition procedure eliminates the last hurdle to perform virtually any kind of imaginable targeted BAC modifications in E. coli. The herpesviral terminal genomic junction was identified as an optimal mini-F vector integration site for the construction of an infectious BAC, which allows the rapid generation of mutant virus without any unwanted secondary genome alterations. The novel mini-F transposition technique can be a valuable tool to optimize, repair or restructure other established BACs as well and may facilitate the development of gene therapy or vaccine vectors

Comparison of homologous and heterologous prime-boost vaccine approaches using Modified Vaccinia Ankara and soluble protein to induce neutralizing antibodies by the human cytomegalovirus pentamer complex in mice

Since neutralizing antibodies (NAb) targeting the human cytomegalovirus (HCMV) pentamer complex (PC) potently block HCMV host cell entry, anti-PC NAb induction is thought to be important for a vaccine formulation to prevent HCMV infection. By developing a vaccine strategy based on soluble PC protein and using a previously generated Modified Vaccinia Ankara vector co-expressing all five PC subunits (MVA-PC), we compared HCMV NAb induction by homologous immunization using prime-boost vaccine regimen employing only PC protein or MVA-PC and heterologous immunization using prime-boost combinations of PC protein and MVA-PC. Utilizing a recently isolated anti-PC NAb, we produced highly pure soluble PC protein that displayed conformational and linear neutralizing epitopes, interfered with HCMV entry, and was recognized by antibodies induced by HCMV during natural infection. Mice vaccinated by different immunization routes with the purified PC protein in combination with a clinically approved adjuvant formulation elicited high-titer and durable HCMV NAb. While MVA-PC and soluble PC protein either alone or in combination elicited robust HCMV NAb, significantly different potencies of these vaccine approaches were observed in dependence on immunization schedule. Using only two immunizations, vaccination with MVA-PC alone or prime-boost combinations of MVA-PC and PC protein was significantly more effective in stimulating HCMV NAb than immunization with PC protein alone. In contrast, with three immunizations, NAb induced by soluble PC protein either alone or combined with two boosts of MVA-PC increased to levels that exceeded NAb titer stimulated by MVA-PC alone. These results provide insights into the potency of soluble protein and MVA to elicit NAb by the HCMV PC via homologous and heterologous prime-boost immunization, which may contribute to develop clinically deployable vaccine strategies to prevent HCMV infection

A Multivalent Kaposi Sarcoma-Associated Herpesvirus-Like Particle Vaccine Capable of Eliciting High Titers of Neutralizing Antibodies in Immunized Rabbits

Kaposi sarcoma-associated herpesvirus (KSHV) is an emerging pathogen and the causative agent of multiple cancers in immunocompromised patients. To date, there is no licensed prophylactic KSHV vaccine. In this study, we generated a novel subunit vaccine that incorporates four key KSHV envelope glycoproteins required for viral entry in diverse cell types (gpK8.1, gB, and gH/gL) into a single multivalent KSHV-like particle (KSHV-LP). Purified KSHV-LPs were similar in size, shape, and morphology to KSHV virions. Vaccination of rabbits with adjuvanted KSHV-LPs generated strong glycoprotein-specific antibody responses, and purified immunoglobulins from KSHV-LP-immunized rabbits neutralized KSHV infection in epithelial, endothelial, fibroblast, and B cell lines (60–90% at the highest concentration tested). These findings suggest that KSHV-LPs may be an ideal platform for developing a safe and effective prophylactic KSHV vaccine. We envision performing future studies in animal models that are susceptible to KSHV infection, to determine correlates of immune protection in vivo

Kaposi Sarcoma-Associated Herpesvirus Glycoprotein H Is Indispensable for Infection of Epithelial, Endothelial, and Fibroblast Cell Types

Kaposi sarcoma-associated herpesvirus (KSHV) is an emerging pathogen and is the causative infectious agent of Kaposi sarcoma and two malignancies of B cell origin. To date, there is no licensed KSHV vaccine. Development of an effective vaccine against KSHV continues to be limited by a poor understanding of how the virus initiates acute primary infection in vivo in diverse human cell types. The role of glycoprotein H (gH) in herpesvirus entry mechanisms remains largely unresolved. To characterize the requirement for KSHV gH in the viral life cycle and in determination of cell tropism, we generated and characterized a mutant KSHV in which expression of gH was abrogated. Using a bacterial artificial chromosome containing a complete recombinant KSHV genome and recombinant DNA technology, we inserted stop codons into the gH coding region. We used electron microscopy to reveal that the gH-null mutant virus assembled and exited from cells normally, compared to wild-type virus. Using purified virions, we assessed infectivity of the gH-null mutant in diverse mammalian cell types in vitro. Unlike wild-type virus or a gH-containing revertant, the gH-null mutant was unable to infect any of the epithelial, endothelial, or fibroblast cell types tested. However, its ability to infect B cells was equivocal and remains to be investigated in vivo due to generally poor infectivity in vitro. Together, these results suggest that gH is critical for KSHV infection of highly permissive cell types, including epithelial, endothelial, and fibroblast cells. IMPORTANCE All homologues of herpesvirus gH studied to date have been implicated in playing an essential role in viral infection of diverse permissive cell types. However, the role of gH in the mechanism of KSHV infection remains largely unresolved. In this study, we generated a gH-null mutant KSHV and provided evidence that deficiency of gH expression did not affect viral particle assembly or egress. Using the gH-null mutant, we showed that gH was indispensable for KSHV infection of epithelial, endothelial, and fibroblast cells in vitro. This suggests that gH is an important target for the development of a KSHV prophylactic vaccine to prevent initial viral infection

Kaposi Sarcoma-associated Herpesvirus Glycoprotein H is Indispensable for Infection of Epithelial, Endothelial, and Fibroblast Cell Types

Kaposi sarcoma-associated herpesvirus (KSHV) is an emerging pathogen and is the causative infectious agent of Kaposi sarcoma and two malignancies of B cell origin. To date, there is no licensed KSHV vaccine. Development of an effective vaccine against KSHV continues to be limited by a poor understanding of how the virus initiates acute primary infection in vivo in diverse human cell types. The role of glycoprotein H (gH) in herpesvirus entry mechanisms remains largely unresolved. To characterize the requirement for KSHV gH in the viral life cycle and in determination of cell tropism, we generated and characterized a mutant KSHV in which expression of gH was abrogated. Using a bacterial artificial chromosome containing a complete recombinant KSHV genome and recombinant DNA technology, we inserted stop codons into the gH coding region. We used electron microscopy to reveal that the gH-null mutant virus assembled and exited from cells normally, compared to wild-type virus. Using purified virions, we assessed infectivity of the gH-null mutant in diverse mammalian cell types in vitro Unlike wild-type virus or a gH-containing revertant, the gH-null mutant was unable to infect any of the epithelial, endothelial, or fibroblast cell types tested. However, its ability to infect B cells was equivocal, and remains to be investigated in vivo due to generally poor infectivity in vitro Together, these results suggest that gH is critical for KSHV infection of highly permissive cell types including epithelial, endothelial, and fibroblasts. MPORTANCE: All homologues of herpesvirus gH studied to date have been implicated in playing an essential role in viral infection of diverse permissive cell types. However, the role of gH in the mechanism of KSHV infection remains largely unresolved. In this study, we generated a gH-null mutant KSHV and provided evidence that deficiency of gH expression did not affect viral particle assembly or egress. Using the gH-null mutant, we showed that gH was indispensable for KSHV infection of epithelial, endothelial, and fibroblast cells in vitro. This suggests that gH is an important target for the development of a KSHV prophylactic vaccine to prevent initial viral infection

Neutralization of Human Cytomegalovirus Entry into Fibroblasts and Epithelial Cells

Human cytomegalovirus (HCMV) is a leading cause of permanent birth defects, highlighting the need to develop an HCMV vaccine candidate. However, HCMV vaccine development is complicated by the varying capacity of neutralizing antibodies (NAb) to interfere in vitro with the HCMV entry routes mediating infection of fibroblast (FB) and epithelial cells (EC). While HCMV infection of FB and EC requires glycoprotein complexes composed of gB and gH/gL/gO, EC infection depends additionally on the envelope pentamer complex (PC) composed of gH, gL, UL128, UL130 and UL131A. Unlike NAb to gB or gH epitopes that can interfere with both FB and EC infection, NAb targeting predominantly conformational epitopes of the UL128/130/131A subunits are unable to prevent FB entry, though they are highly potent in blocking EC infection. Despite the selective requirement of the PC for EC entry, the PC is exceptionally immunogenic as vaccine antigen to stimulate both EC- and FB-specific NAb responses due to its capacity to elicit NAb that target epitopes of the UL128/130/131A subunits and gH. These findings suggest that the PC could be sufficient in a subunit vaccine formulation to induce robust FB- and EC-specific NAb responses. In this short review, we discuss NAb responses induced through natural infection and vaccination that interfere in vitro with HCMV infection of FB and EC