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Human cytomegalovirus (CMV) is the most common infectious cause of complications post-transplantation, while a CMV vaccine for transplant recipients has yet to be licensed. Triplex, a multiantigen Modified Vaccinia Ankara (MVA)-vectored CMV vaccine candidate based on the immunodominant antigens phosphoprotein 65 (pp65) and immediate-early 1 and 2 (IE1/2), is in an advanced stage of clinical development. However, its limited genetic and expression stability restricts its potential for large-scale production. Using a recently developed fully synthetic MVA (sMVA) platform, we developed a new generation Triplex vaccine candidate, T10-F10, with different sequence modifications for enhanced vaccine stability. T10-F10 demonstrated genetic and expression stability during extensive virus passaging. In addition, we show that T10-F10 confers comparable immunogenicity to the original Triplex vaccine to elicit antigen-specific T cell responses in HLA-transgenic mice. These results demonstrate improvements in translational vaccine properties of an sMVA-based CMV vaccine candidate designed as a therapeutic treatment for transplant recipients., (© 2024. The Author(s).)

Background: Although the mpox global health emergency caused by mpox virus (MPXV) clade IIb.1 has ended, mpox cases are still reported due to low vaccination coverage and waning immunity. COH04S1 is a clinically evaluated, multiantigen COVID-19 vaccine candidate built on a fully synthetic platform of the highly attenuated modified vaccinia Ankara (MVA) vector, representing the only FDA-approved smallpox/mpox vaccine JYNNEOS. Given the potential threat of MPXV resurgence and need for vaccine alternatives, we aimed to assess the capacity COH04S1 and its synthetic MVA (sMVA) backbone to confer MPXV-specific immunity., Methods: We evaluated orthopoxvirus-specific and MPXV cross-reactive immune responses in samples collected during a Phase 1 clinical trial of COH04S1 and in non-human primates (NHP) vaccinated with COH04S1 or its sMVA backbone. MPXV cross-reactive immune responses in COH04S1-vaccinated healthy adults were compared to responses measured in healthy subjects vaccinated with JYNNEOS. Additionally, we evaluated the protective efficacy of COH04S1 and sMVA against mpox in mpox-susceptible CAST/EiJ mice., Results: COH04S1-vaccinated individuals develop robust orthopoxvirus-specific humoral and cellular responses, including cross-reactive antibodies to MPXV-specific virion proteins as well as MPXV cross-neutralizing antibodies in 45% of the subjects. In addition, NHP vaccinated with COH04S1 or sMVA show similar MPXV cross-reactive antibody responses. Moreover, MPXV cross-reactive humoral responses elicited by COH04S1 are comparable to those measured in JYNNEOS-vaccinated subjects. Finally, we show that mice vaccinated with COH04S1 or sMVA are protected from lung infection following challenge with MPXV clade IIb.1., Conclusions: These results demonstrate the capacity of sMVA vaccines to elicit cross-reactive and protective orthopox-specific immunity against MPXV, suggesting that COH04S1 and sMVA could be developed as bivalent or monovalent mpox vaccine alternatives against MPXV., (© 2024. The Author(s).)

Competing Interests: FC and FW received royalty payments from Helocyte related to a CMV vaccine. FC and FW hold a patent on CMV neutralising antibodies. FW holds a patent on CMV vaccine construction.

Hematopoietic cell transplantation (HCT) and chimeric antigen receptor (CAR)-T cell patients are immunocompromised, remain at high risk following SARS-CoV-2 infection, and are less likely than immunocompetent individuals to respond to vaccination. As part of the safety lead-in portion of a phase 2 clinical trial in patients post HCT/CAR-T for hematological malignancies (HM), we tested the immunogenicity of the synthetic modified vaccinia Ankara-based COVID-19 vaccine COH04S1 co-expressing spike (S) and nucleocapsid (N) antigens. Thirteen patients were vaccinated 3-12 months post HCT/CAR-T with two to four doses of COH04S1. SARS-CoV-2 antigen-specific humoral and cellular immune responses, including neutralizing antibodies to ancestral virus and variants of concern (VOC), were measured up to six months post vaccination and compared to immune responses in historical cohorts of naïve healthy volunteers (HV) vaccinated with COH04S1 and naïve healthcare workers (HCW) vaccinated with the FDA-approved mRNA vaccine Comirnaty ® (Pfizer, New York, NY, USA). After one or two COH04S1 vaccine doses, HCT/CAR-T recipients showed a significant increase in S- and N-specific binding antibody titers and neutralizing antibodies with potent activity against SARS-CoV-2 ancestral virus and VOC, including the highly immune evasive Omicron XBB.1.5 variant. Furthermore, vaccination with COH04S1 resulted in a significant increase in S- and N-specific T cells, predominantly CD4 + T lymphocytes. Elevated S- and N-specific immune responses continued to persist at six months post vaccination. Furthermore, both humoral and cellular immune responses in COH04S1-vaccinated HCT/CAR-T patients were superior or comparable to those measured in COH04S1-vaccinated HV or Comirnaty ® -vaccinated HCW. These results demonstrate robust stimulation of SARS-CoV-2 S- and N-specific immune responses including cross-reactive neutralizing antibodies by COH04S1 in HM patients post HCT/CAR-T, supporting further testing of COH04S1 in immunocompromised populations.

Emerging SARS-CoV-2 Omicron subvariants continue to disrupt COVID-19 vaccine efficacy through multiple immune mechanisms including neutralizing antibody evasion. We developed COH04S1, a synthetic modified vaccinia Ankara vector that co-expresses Wuhan-Hu-1-based spike and nucleocapsid antigens. COH04S1 demonstrated efficacy against ancestral virus and Beta and Delta variants in animal models and was safe and immunogenic in a Phase 1 clinical trial. Here, we report efficacy of COH04S1 and analogous Omicron BA.1- and Beta-specific vaccines to protect Syrian hamsters from Omicron subvariants. Despite eliciting strain-specific antibody responses, all three vaccines protect hamsters from weight loss, lower respiratory tract infection, and lung pathology following challenge with Omicron BA.1 or BA.2.12.1. While the BA.1-specifc vaccine affords consistently improved efficacy compared to COH04S1 to protect against homologous challenge with BA.1, all three vaccines confer similar protection against heterologous challenge with BA.2.12.1. These results demonstrate efficacy of COH04S1 and variant-specific derivatives to confer cross-protective immunity against SARS-CoV-2 Omicron subvariants., (© 2023. The Author(s).)

Cell-mediated immunity may contribute to providing protection against SARS-CoV-2 and its variants of concern (VOC). We developed COH04S1, a synthetic multiantigen modified vaccinia Ankara (MVA)-based COVID-19 vaccine that stimulated potent spike (S) and nucleocapsid (N) antigen-specific humoral and cellular immunity in a phase 1 clinical trial in healthy adults. Here, we show that individuals vaccinated with COH04S1 or mRNA vaccine BNT162b2 maintain robust cross-reactive cellular immunity for six or more months post-vaccination. Although neutralizing antibodies induced in COH04S1- and BNT162b2-vaccinees showed reduced activity against Delta and Omicron variants compared to ancestral SARS-CoV-2, S-specific T cells elicited in both COH04S1- and BNT162b2-vaccinees and N-specific T cells elicited in COH04S1-vaccinees demonstrated potent and equivalent cross-reactivity against ancestral SARS-CoV-2 and the major VOC. These results suggest that vaccine-induced T cells to S and N antigens may constitute a critical second line of defense to provide long-term protection against SARS-CoV-2 VOC., Competing Interests: While unknown whether the publication of this report will aid in receiving grants and contracts, it is possible that this publication will be of benefit to the City of Hope (COH). COH had no role in the conceptualization, design, data collection, analysis, decision to publish, or preparation of the article. DJD and FW are co-inventors on a patent application covering the design and construction of the synthetic MVA platform (PCT/US2021/016,247). DJD, FW, and FC are co-inventors on a patent application covering the development of a COVID-19 vaccine (PCT/US2021/032,821). DJD is a consultant for GeoVax. All other authors declare no competing interests. GeoVax Labs Inc. has taken a worldwide exclusive license for COH04S1 under the name of GEO-CM04S1., (© 2022 The Author(s).)

COVID-19 vaccine efficacy is threatened by emerging SARS-CoV-2 variants of concern (VOC) with the capacity to evade protective neutralizing antibody responses. We recently developed clinical vaccine candidate COH04S1, a synthetic modified vaccinia Ankara vector (sMVA) co-expressing spike and nucleocapsid antigens based on the Wuhan-Hu-1 reference strain that showed potent efficacy to protect against ancestral SARS-CoV-2 in Syrian hamsters and non-human primates and was safe and immunogenic in healthy volunteers. Here, we demonstrate that intramuscular immunization of Syrian hamsters with COH04S1 and an analogous Beta variant-adapted vaccine candidate (COH04S351) elicits potent cross-reactive antibody responses and protects against weight loss, lower respiratory tract infection, and lung pathology following challenge with major SARS-CoV-2 VOC, including Beta and the highly contagious Delta variant. These results demonstrate efficacy of COH04S1 and a variant-adapted vaccine analog to confer cross-protective immunity against SARS-CoV-2 and its emerging VOC, supporting clinical investigation of these sMVA-based COVID-19 vaccine candidates., Competing Interests: While unknown whether publication of this report will aid in receiving grants and contracts, it is possible that this publication will be of benefit to City of Hope (COH). COH had no role in the conceptualization, design, data collection, analysis, decision to publish, or preparation of the manuscript. D.J.D. and F.W. are co-inventors on patent application PCT/US2021/016247 which covers the design and construction of the synthetic MVA platform. D.J.D., F.W., and F.C. are co-inventors on PCT/US2021/032821 and 63/244,103 which cover the development of a COVID-19 vaccine. D.J.D. is a consultant for GeoVax Labs Inc. All remaining authors declare no competing interests. GeoVax Labs Inc. has taken a worldwide exclusive license for COH04S1 from COH., (© 2022 The Author(s).)

Background: COH04S1, a synthetic attenuated modified vaccinia virus Ankara vector co-expressing SARS-CoV-2 spike and nucleocapsid antigens, was tested for safety and immunogenicity in healthy adults., Methods: This combined open-label and randomised, phase 1 trial was done at the City of Hope Comprehensive Cancer Center (Duarte, CA, USA). We included participants aged 18-54 years with a negative SARS-CoV-2 antibody and PCR test, normal haematology and chemistry panels, a normal electrocardiogram and troponin concentration, negative pregnancy test if female, body-mass index of 30 kg/m 2 or less, and no modified vaccinia virus Ankara or poxvirus vaccine in the past 12 months. In the open-label cohort, 1·0 × 10 7 plaque-forming units (PFU; low dose), 1·0 × 10 8 PFU (medium dose), and 2·5 × 10 8 PFU (high dose) of COH04S1 were administered by intramuscular injection on day 0 and 28 to sentinel participants using a queue-based statistical design to limit risk. In a randomised dose expansion cohort, additional participants were randomly assigned (3:3:1), using block size of seven, to receive two placebo vaccines (placebo group), one low-dose COH04S1 and one placebo vaccine (low-dose COH04S1 plus placebo group), or two low-dose COH04S1 vaccines (low-dose COH04S1 group). The primary outcome was safety and tolerability, with secondary objectives assessing vaccine-specific immunogenicity. The primary immunological outcome was a four times increase (seroconversion) from baseline in spike-specific or nucleocapsid-specific IgG titres within 28 days of the last injection, and seroconversion rates were compared with participants who received placebo using Fisher's exact test. Additional secondary outcomes included assessment of viral neutralisation and cellular responses. This trial is registered with ClinicalTrials.gov, NCT046339466., Findings: Between Dec 13, 2020, and May 24, 2021, 56 participants initiated vaccination. On day 0 and 28, 17 participants received low-dose COH04S1, eight received medium-dose COH04S1, nine received high-dose COH04S1, five received placebo, 13 received low-dose COH04S1 followed by placebo, and four discontinued early. Grade 3 fever was observed in one participant who received low-dose COH04S1 and placebo, and grade 2 anxiety or fatigue was seen in one participant who received medium-dose COH04S1. No severe adverse events were reported. Seroconversion was observed in all 34 participants for spike protein and 32 (94%) for nucleocapsid protein (p<0·0001 vs placebo for each comparison). Four times or more increase in SARS-CoV-2 neutralising antibodies within 56 days was measured in nine of 17 participants in the low-dose COH04S1 group, all eight participants in the medium-dose COH04S1 group, and eight of nine participants in the high-dose COH04S1 group (p=0·0035 combined dose levels vs placebo). Post-prime and post-boost four times increase in spike-specific or nucleocapsid-specific T cells secreting interferon-γ was measured in 48 (98%; 95% CI 89-100) of 49 participants who received at least one dose of COH04S1 and provided a sample for immunological analysis., Interpretation: COH04S1 was well tolerated and induced spike-specific and nucleocapsid-specific antibody and T-cell responses. Future evaluation of this COVID-19 vaccine candidate as a primary or boost vaccination is warranted., Funding: The Carol Moss Foundation and City of Hope Integrated Drug Development Venture programme., Competing Interests: DJD and FW are co-inventors on a patent application covering the design and construction of the synthetic modified vaccinia Ankara platform (PCT/US2021/016247). DJD, FW, and FC are co-inventors on a patent application covering the development of a COVID-19 vaccine (PCT/US2021/032821). FC, JAZ, PHF, RS, JD, BW, AMA, KFr, RAT, JKD, SD, AGP, DDN, PA, YC, HC, CLR, KT, YP, JM, AI, QZ, VK, DJ, KFa, TK, JN, MK, VHN, SOF, AW, FW, and DJD are employees of City of Hope National Medical Center (Duarte, CA, USA), which developed the vaccine and funded the trial. AS is a consultant for Gritstone, Flow Pharma, Merck, Epitogenesis, Gilead, and Avalia. La Jolla Institute for Immunology has filed for patent protection for various aspects of T-cell epitope and vaccine design work. All other authors declare no competing interests., (© 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license.)

Second-generation COVID-19 vaccines could contribute to establish protective immunity against SARS-CoV-2 and its emerging variants. We developed COH04S1, a synthetic multiantigen modified vaccinia Ankara-based SARS-CoV-2 vaccine that co-expresses spike and nucleocapsid antigens. Here, we report COH04S1 vaccine efficacy in animal models. We demonstrate that intramuscular or intranasal vaccination of Syrian hamsters with COH04S1 induces robust Th1-biased antigen-specific humoral immunity and cross-neutralizing antibodies (NAb) and protects against weight loss, lower respiratory tract infection, and lung injury following intranasal SARS-CoV-2 challenge. Moreover, we demonstrate that single-dose or two-dose vaccination of non-human primates with COH04S1 induces robust antigen-specific binding antibodies, NAb, and Th1-biased T cells, protects against both upper and lower respiratory tract infection following intranasal/intratracheal SARS-CoV-2 challenge, and triggers potent post-challenge anamnestic antiviral responses. These results demonstrate COH04S1-mediated vaccine protection in animal models through different vaccination routes and dose regimens, complementing ongoing investigation of this multiantigen SARS-CoV-2 vaccine in clinical trials., (© 2022. The Author(s).)

Introduction: The aim of this study was to determine the incidence and risk factors for postoperative flank bulging and associated physiologic/psychologic consequences as well as to establish a clinical flank bulge classification system after open retroperitoneal surgery., Methods: In this retrospective study, a postal patient survey was sent to 240 patients who underwent open retroperitoneal surgery between 2007 and 2017. Patients, who reported a flank bulge, were invited for further evaluation, which included a clinical examination and standardized photo documentation., Results: Forty-three of 120 patients (35.8%) reported a flank bulging after retroperitoneal surgery. During clinical examination, a flank bulge could be confirmed in 25 patients, whereas in 18 patients, no bulging could be detected, leading to a corrected rate of flank bulge-positive patients of 20.8%. The corresponding relaxation values ranged from 1 to 1.44 and correlated with the clinical degree of bulging. A body mass index of ≥25 was identified as a risk factor. No correlation was found regarding age, gender, surgery side, access to the retroperitoneum, surgical procedure, and pathology. Thirty-seven patients complained about chronic pain or suffered from the cosmetic impact of bulging. Thirteen of those patients had shown a flank bulge during clinical examination, resulting in a symptomatic bulge rate of 10.8% (13/120 patients)., Conclusion: Chronic pain and postoperative flank bulging are 2 of the most common long-term complications after open retroperitoneal access. If an open retroperitoneal approach is required, particularly obese patients should be thoroughly informed about the risk of flank bulging and chronic pain., (© 2021 S. Karger AG, Basel.)

Maternal reinfection of immune women with novel human cytomegalovirus (HCMV) strains acquired during pregnancy can result in symptomatic congenital CMV (cCMV) infection. Novel animal model strategies are needed to explore vaccine-mediated protections against maternal reinfection. To investigate this in the guinea pig cytomegalovirus (GPCMV) model, a strictly in vivo-passaged workpool of a novel strain, the CIDMTR strain (dose, 1 × 10 7 pfu) was used to infect dams that had been challenged in a previous pregnancy with the 22122 strain, following either sham-immunization (vector only) or vaccination with MVA-vectored gB, gH/gL, or pentameric complex (PC) vaccines. Maternal DNAemia cleared by day 21 in the glycoprotein-vaccinated dams, but not in the sham-immunized dams. Mean pup birth weights were 72.85 ± 10.2, 80.0 ± 6.9, 81.4 ± 14.1, and 89.38 ± 8.4 g in sham-immunized, gB, gH/gL, and PC groups, respectively ( p < 0.01 for control v. PC). Pup mortality in the sham-immunized group was 6/12 (50%), but reduced to 3/35 (8.6%) in combined vaccine groups ( p = 0.0048). Vertical CIDMTR transmission occurred in 6/12 pups (50%) in the sham-vaccinated group, compared to 2/34 pups (6%) in the vaccine groups ( p = 0.002). We conclude that guinea pigs immunized with vectored vaccines expressing 22122 strain-specific glycoproteins are protected after a reinfection with a novel, heterologous clinical isolate (CIDMTR) in a second pregnancy.

Modified Vaccinia Ankara (MVA) is a highly attenuated poxvirus vector that is widely used to develop vaccines for infectious diseases and cancer. We demonstrate the construction of a vaccine platform based on a unique three-plasmid system to efficiently generate recombinant MVA vectors from chemically synthesized DNA. In response to the ongoing global pandemic caused by SARS coronavirus-2 (SARS-CoV-2), we use this vaccine platform to rapidly produce fully synthetic MVA (sMVA) vectors co-expressing SARS-CoV-2 spike and nucleocapsid antigens, two immunodominant antigens implicated in protective immunity. We show that mice immunized with these sMVA vectors develop robust SARS-CoV-2 antigen-specific humoral and cellular immune responses, including potent neutralizing antibodies. These results demonstrate the potential of a vaccine platform based on synthetic DNA to efficiently generate recombinant MVA vectors and to rapidly develop a multi-antigenic poxvirus-based SARS-CoV-2 vaccine candidate.

Human cytomegalovirus (CMV) is a ubiquitous pathogen that causes significant morbidity in some vulnerable populations. Individualized adoptive transfer of ex vivo expanded CMV-specific CD8+ T cells has provided proof-of-concept that immunotherapy can be highly effective, but a chimeric antigen receptor (CAR) approach would provide a feasible method for broad application. We created 8 novel CARs using anti-CMV neutralizing antibody sequences, which were transduced via lentiviral vector into primary CD8+ T cells. All CARs were expressed. Activity against CMV-infected target cells was assessed by release of cytokines (interferon-γ and tumor necrosis factor-α), upregulation of surface CD107a, proliferation, cytolysis of infected cells, and suppression of viral replication. While some CARs showed varying functional activity across these assays, 1 CAR based on antibody 21E9 was consistently superior in all measures. These results support development of a CMV-specific CAR for therapeutic use against CMV and potentially other applications harnessing CMV-driven immunotherapies., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Although congenital infection by human cytomegalovirus (HCMV) is well recognized as a leading cause of neurodevelopmental defects, HCMV neuropathogenesis remains poorly understood. A major challenge for investigating HCMV-induced abnormal brain development is the strict CMV species specificity, which prevents the use of animal models to directly study brain defects caused by HCMV. We show that infection of human-induced pluripotent-stem-cell-derived brain organoids by a "clinical-like" HCMV strain results in reduced brain organoid growth, impaired formation of cortical layers, and abnormal calcium signaling and neural network activity. Moreover, we show that the impeded brain organoid development caused by HCMV can be prevented by neutralizing antibodies (NAbs) that recognize the HCMV pentamer complex. These results demonstrate in a three-dimensional cellular biosystem that HCMV can impair the development and function of the human brain and provide insights into the potential capacity of NAbs to mitigate brain defects resulted from HCMV infection., Competing Interests: F.W., F.C., and D.J.D. receive royalty payments from Helocyte Inc. D.J.D. chairs the Scientific Advisory Board of Helocyte Inc., and he has an equity interest in Helocyte Inc. F.W., F.C., and D.J.D. hold patents US10487139B2 and US10376575B2., (© 2020 The Authors.)

: The use of cytomegalovirus (CMV) as a vaccine vector to express antigens against multiple infectious diseases, including simian immunodeficiency virus, Ebola virus, plasmodium, and mycobacterium tuberculosis, in rhesus macaques has generated extraordinary levels of protective immunity against subsequent pathogenic challenge. Moreover, the mechanisms of immune protection have altered paradigms about viral vector-mediated immunity against ectopically expressed vaccine antigens. Further optimization of CMV-vectored vaccines, particularly as this approach moves to human clinical trials will be augmented by a more complete understanding of how CMV engenders mechanisms of immune protection. This review summarizes the particulars of the specific CMV vaccine vector that has been used to date (rhesus CMV strain 68-1) in relation to CMV natural history.

(1) Background: A congenital cytomegalovirus (cCMV) vaccine is a major research priority, but the essential glycoprotein target(s) remain unclear. We compared CMV gB (gpgB), gH/gL (gp75/gL), and pentameric complex (gpPC, composed of gH/gL/GP129/GP131/GP133) vaccines in a guinea pig CMV (GPCMV) congenital infection model. (2) Methods: Modified vaccinia virus Ankara (MVA) vaccines expressing GPCMV glycoproteins were used to immunize GPCMV-seronegative, female Hartley guinea pigs (three-dose series, 3 × 10 7 pfu/dose). After pregnancy was established, the dams underwent an early third-trimester challenge with salivary gland (SG)-adapted GPCMV. (3) Results: All vaccines elicited GPCMV-specific binding and neutralizing antibodies. Preconception immunization resulted in 19.5-, 4.9-, and 698-fold reductions in maternal DNAemia in MVA-gp75/gL, MVA-gpPC and MVA-gpgB groups, respectively, at day 14, post-SG challenge. Vaccination improved pups' birth weight and reduced mortality and congenital CMV transmission. In controls, cCMV infection was observed in 100% of pups (mean viral load in all visceral organs, 2.4 × 10 4 genomes/mg), versus 50% in the gB group (visceral viral load, 9.4 × 10 2 genomes/mg; p < 0.05). No significant reductions in congenital transmission were noted in the MVA-gp75/gL and MVA-gpPC groups. (4) Conclusions: MVA-vectored gB, gH/gL, and PC vaccines were immunogenic, and protected against maternal DNAemia and pup mortality. These results support the inclusion of multiple glycoprotein complexes in a cCMV vaccine.

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., (Copyright © 2019 American Society for Microbiology.)

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., (Copyright © 2019. Published by Elsevier Ltd.)

Introduction: It has been almost fifty years since the Towne strain was used by Plotkin and collaborators as the first vaccine candidate for cytomegalovirus (CMV). While that approach showed partial efficacy, there have been a multitude of challenges to improve on the promise of a CMV vaccine. Efforts have been dichotomized into a therapeutic vaccine for patients with CMV-infected allografts, either stem cells or solid organ, and a prophylactic vaccine for congenital infection., Areas Covered: This review will evaluate research prospects for a therapeutic vaccine for transplant recipients that recognizes CMV utilizing primarily T cell responses. Similarly, we will provide an extensive discussion on attempts to develop a vaccine to prevent the manifestations of congenital infection, based on eliciting a humoral anti-CMV protective response. The review will also describe newer developments that have upended the efforts toward such a vaccine through the discovery of a second pathway of CMV infection that utilizes an alternative receptor for entry using a series of antigens that have been determined to be important for prevention of infection., Expert Commentary: There is a concerted effort to unify separate therapeutic and prophylactic vaccine strategies into a single delivery agent that would be effective for both transplant-related and congenital infection.

As human cytomegalovirus (HCMV) is a common cause of disease in newborns and transplant recipients, developing an HCMV vaccine is considered a major public health priority. Yet an HCMV vaccine candidate remains elusive. Although the precise HCMV immune correlates of protection are unclear, both humoral and cellular immune responses have been implicated in protection against HCMV infection and disease. Here we describe a vaccine approach based on the well-characterized modified vaccinia virus Ankara (MVA) vector to stimulate robust HCMV humoral and cellular immune responses by an antigen combination composed of the envelope pentamer complex (PC), glycoprotein B (gB), and phosphoprotein 65 (pp65). We show that in mice, multiantigenic MVA vaccine vectors simultaneously expressing all five PC subunits, gB, and pp65 elicit potent complement-independent and complement-dependent HCMV neutralizing antibodies as well as mouse and human MHC-restricted, polyfunctional T cell responses by the individual antigens. In addition, we demonstrate that the PC/gB antigen combination of these multiantigenic MVA vectors can enhance the stimulation of humoral immune responses that mediate in vitro neutralization of different HCMV strains and antibody-dependent cellular cytotoxicity. These results support the use of MVA to develop a multiantigenic vaccine candidate for controlling HCMV infection and disease in different target populations, such as pregnant women and transplant recipients. IMPORTANCE The development of a human cytomegalovirus (HCMV) vaccine to prevent congenital disease and transplantation-related complications is an unmet medical need. While many HCMV vaccine candidates have been developed, partial success in preventing or controlling HCMV infection in women of childbearing age and transplant recipients has been observed with an approach based on envelope glycoprotein B (gB). We introduce a novel vaccine strategy based on the clinically deployable modified vaccinia virus Ankara (MVA) vaccine vector to elicit potent humoral and cellular immune responses by multiple immunodominant HCMV antigens, including gB, phosphoprotein 65, and all five subunits of the pentamer complex. These findings could contribute to development of a multiantigenic vaccine strategy that may afford more protection against HCMV infection and disease than a vaccine approach employing solely gB., (Copyright © 2018 American Society for Microbiology.)

Neutralizing antibodies (NAb) interfering with glycoprotein complex-mediated virus entry into host cells are thought to contribute to the protection against herpesvirus infection. However, using herpesvirus glycoprotein complexes as vaccine antigens can be complicated by the necessity of expressing multiple subunits simultaneously to allow efficient complex assembly and formation of conformational NAb epitopes. By using a novel bacterial artificial chromosome (BAC) clone of the clinically deployable Modified Vaccinia Ankara (MVA) vector and exploiting ribosomal skipping mediated by 2A peptides, MVA vectors were generated that expressed self-processing subunits of the human cytomegalovirus (HCMV) pentamer complex (PC) composed of gH, gL, UL128, UL130, and UL131A. These MVA vectors expressed 2A-linked HCMV PC subunits that were efficiently cleaved and transported to the cell surface as protein complexes forming conformational neutralizing epitopes. In addition, vaccination of mice by only two immunizations with these MVA vectors resulted in potent HCMV NAb responses that remained stable over a period of at least six months. This method of eliciting NAb by 2A-linked, self-processing HCMV PC subunits could contribute to develop a HCMV vaccine candidate and may serve as a template to facilitate the development of subunit vaccine strategies against other herpesviruses., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., Competing Interests: All authors receive research support from Helocyte Inc. Don J. Diamond, Felix Wussow and Flavia Chiuppesi receive royalty payments from Helocyte Inc. Don J. Diamond chairs the Scientific Advisory Board and has an equity interest in Helocyte Inc.

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.

Human cytomegalovirus (HCMV) is the most common congenital infection and a known cause of microcephaly, sensorineural hearing loss, and cognitive impairment among newborns worldwide. Natural maternal HCMV immunity reduces the incidence of congenital infection, but does not prevent the disease altogether. We employed a nonhuman primate model of congenital CMV infection to investigate the ability of preexisting antibodies to protect against placental CMV transmission in the setting of primary maternal infection and subsequent viremia, which is required for placental virus exposure. Pregnant, CD4+ T cell-depleted, rhesus CMV-seronegative (RhCMV-seronegative) rhesus monkeys were treated with either standardly produced hyperimmune globulin (HIG) from RhCMV-seropositive macaques or dose-optimized, potently RhCMV-neutralizing HIG prior to intravenous challenge with an RhCMV mixture. HIG passive infusion provided complete protection against fetal loss in both groups. The dose-optimized, RhCMV-neutralizing HIG additionally inhibited placental transmission of RhCMV and reduced viral replication and diversity. Our findings suggest that the presence of durable and potently neutralizing antibodies at the time of primary infection can prevent transmission of systemically replicating maternal RhCMV to the developing fetus, and therefore should be a primary target of vaccines to eliminate this neonatal infection.

Human cytomegalovirus (HCMV) is the most common congenital infection worldwide and the leading infectious cause of neurologic deficits and hearing loss in newborns. Development of a maternal HCMV vaccine to prevent vertical virus transmission is a high priority, yet protective maternal immune responses following acute infection are poorly understood. To characterize the maternal humoral immune response to primary CMV infection, we investigated the plasmablast and early antibody repertoire using a nonhuman primate model with two acutely rhesus CMV (RhCMV)-infected animals-a CD4 + T cell-depleted dam that experienced fetal loss shortly after vertical RhCMV transmission and an immunocompetent dam that did not transmit RhCMV to her infant. Compared to the CD4 + T cell-depleted dam that experienced fetal loss, the immunocompetent, nontransmitting dam had a more rapid and robust plasmablast response that produced a high proportion of RhCMV-reactive antibodies, including the first identified monoclonal antibody specific for soluble and membrane-associated RhCMV envelope glycoprotein B (gB). Additionally, we noted that plasmablast RhCMV-specific antibodies had variable gene usage and maturation similar to those observed in a monkey chronically coinfected with simian immunodeficiency virus (SIV) and RhCMV. This study reveals characteristics of the early maternal RhCMV-specific humoral immune responses to primary RhCMV infection in rhesus monkeys and may contribute to a future understanding of what antibody responses should be targeted by a vaccine to eliminate congenital HCMV transmission. Furthermore, the identification of an RhCMV gB-specific monoclonal antibody underscores the possibility of modeling future HCMV vaccine strategies in this nonhuman primate model., (Copyright © 2017 American Society for Microbiology.)

As human cytomegalovirus (HCMV) is the most common infectious cause of fetal anomalies during pregnancy, development of a vaccine that prevents HCMV infection is considered a global health priority. Although HCMV immune correlates of protection are only poorly defined, neutralizing antibodies (NAb) targeting the envelope pentamer complex (PC) composed of the subunits gH, gL, UL128, UL130, and UL131A are thought to contribute to the prevention of HCMV infection. Here, we describe a continuous target sequence within UL128 that is recognized by a previously isolated potent PC-specific NAb termed 13B5. By using peptide-based scanning procedures, we identified a 13-amino-acid-long target sequence at the UL128 C terminus that binds the 13B5 antibody with an affinity similar to that of the purified PC. In addition, the 13B5 binding site is universally conserved in HCMV, contains a previously described UL128/gL interaction site, and interferes with the 13B5 neutralizing function, indicating that the 13B5 epitope sequence is located within the PC at a site of critical importance for HCMV neutralization. Vaccination of mice with peptides containing the 13B5 target sequence resulted in the robust stimulation of binding antibodies and, in a subset of immunized animals, in the induction of detectable NAb, supporting that the identified 13B5 target sequence constitutes a PC-specific neutralizing epitope. These findings provide evidence for the discovery of a continuous neutralizing epitope within the UL128 subunit of the PC that could be an important target of humoral immune responses that are involved in protection against congenital HCMV infection. IMPORTANCE Neutralizing antibodies (NAb) targeting the human cytomegalovirus (HCMV) envelope pentamer complex (PC) are thought to be important for preventing HCMV transmission from the mother to the fetus, thereby mitigating severe developmental disabilities in newborns. However, the epitope sequences within the PC that are recognized by these potentially protective antibody responses are only poorly defined. Here, we provide evidence for the existence of a highly conserved, continuous, PC-specific epitope sequence that appears to be located within the PC at a subunit interaction site of critical importance for HCMV neutralization. These discoveries provide insights into a continuous PC-specific neutralizing epitope, which could be an important target for a vaccine formulation to interfere with congenital HCMV infection., (Copyright © 2017 American Society for Microbiology.)

Attenuated poxvirus modified vaccinia Ankara (MVA) is a useful viral-based vaccine for clinical investigation, because of its excellent safety profile and property of inducing potent immune responses against recombinant (r) antigens. We developed Triplex by constructing an rMVA encoding 3 immunodominant cytomegalovirus (CMV) antigens, which stimulates a host antiviral response: UL83 (pp65), UL123 (IE1-exon4), and UL122 (IE2-exon5). We completed the first clinical evaluation of the Triplex vaccine in 24 healthy adults, with or without immunity to CMV and vaccinia virus (previous DryVax smallpox vaccination). Three escalating dose levels (DL) were administered IM in 8 subjects/DL, with an identical booster injection 28 days later and 1-year follow-up. Vaccinations at all DL were safe with no dose-limiting toxicities. No vaccine-related serious adverse events were documented. Local and systemic reactogenicity was transient and self-limiting. Robust, functional, and durable Triplex-driven expansions of CMV-specific T cells were detected by measuring T-cell surface levels of 4-1BB (CD137), binding to CMV-specific HLA multimers, and interferon-γ production. Marked and durable CMV-specific T-cell responses were also detected in Triplex-vaccinated CMV-seronegatives, and in DryVax-vaccinated subjects. Long-lived memory effector phenotype, associated with viral control during CMV primary infection, was predominantly found on the membrane of CMV-specific and functional T cells, whereas off-target vaccine responses activating memory T cells from the related herpesvirus Epstein-Barr virus remained undetectable. Combined safety and immunogenicity results of MVA in allogeneic hematopoietic stem cell transplant (HCT) recipients and Triplex in healthy adults motivated the initiation of a placebo-controlled multicenter trial of Triplex in HCT patients. This trial was registered at www.clinicaltrials.gov as #NCT02506933., (© 2017 by The American Society of Hematology.)

Unlabelled: Human cytomegalovirus (HCMV) elicits neutralizing antibodies (NAb) of various potencies and cell type specificities to prevent HCMV entry into fibroblasts (FB) and epithelial/endothelial cells (EpC/EnC). NAb targeting the major essential envelope glycoprotein complexes gB and gH/gL inhibit both FB and EpC/EnC entry. In contrast to FB infection, HCMV entry into EpC/EnC is additionally blocked by extremely potent NAb to conformational epitopes of the gH/gL/UL128/130/131A pentamer complex (PC). We recently developed a vaccine concept based on coexpression of all five PC subunits by a single modified vaccinia virus Ankara (MVA) vector, termed MVA-PC. Vaccination of mice and rhesus macaques with MVA-PC resulted in a high titer and sustained NAb that blocked EpC/EnC infection and lower-titer NAb that inhibited FB entry. However, antibody function responsible for the neutralizing activity induced by the MVA-PC vaccine is uncharacterized. Here, we demonstrate that MVA-PC elicits NAb with cell type-specific neutralization potency and antigen recognition pattern similar to human NAb targeting conformational and linear epitopes of the UL128/130/131A subunits or gH. In addition, we show that the vaccine-derived PC-specific NAb are significantly more potent than the anti-gH NAb to prevent HCMV spread in EpC and infection of human placental cytotrophoblasts, cell types thought to be of critical importance for HCMV transmission to the fetus. These findings further validate MVA-PC as a clinical vaccine candidate to elicit NAb that resembles those induced during HCMV infection and provide valuable insights into the potency of PC-specific NAb to interfere with HCMV cell-associated spread and infection of key placental cells., Importance: As a consequence of the leading role of human cytomegalovirus (HCMV) in causing permanent birth defects, developing a vaccine against HCMV has been assigned a major public health priority. We have recently introduced a vaccine strategy based on a widely used, safe, and well-characterized poxvirus vector platform to elicit potent and durable neutralizing antibody (NAb) responses targeting the HCMV envelope pentamer complex (PC), which has been suggested as a critical component for a vaccine to prevent congenital HCMV infection. With this work, we confirm that the NAb elicited by the vaccine vector have properties that are similar to those of human NAb isolated from individuals chronically infected with HCMV. In addition, we show that PC-specific NAb have potent ability to prevent infection of key placental cells that HCMV utilizes to cross the fetal-maternal interface, suggesting that NAb targeting the PC may be essential to prevent HCMV vertical transmission., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Elucidation of maternal immune correlates of protection against congenital cytomegalovirus (CMV) is necessary to inform future vaccine design. Here, we present a novel rhesus macaque model of placental rhesus CMV (rhCMV) transmission and use it to dissect determinants of protection against congenital transmission following primary maternal rhCMV infection. In this model, asymptomatic intrauterine infection was observed following i.v. rhCMV inoculation during the early second trimester in two of three rhCMV-seronegative pregnant females. In contrast, fetal loss or infant CMV-associated sequelae occurred in four rhCMV-seronegative pregnant macaques that were CD4(+) T-cell depleted at the time of inoculation. Animals that received the CD4(+) T-cell-depleting antibody also exhibited higher plasma and amniotic fluid viral loads, dampened virus-specific CD8(+) T-cell responses, and delayed production of autologous neutralizing antibodies compared with immunocompetent monkeys. Thus, maternal CD4(+) T-cell immunity during primary rhCMV infection is important for controlling maternal viremia and inducing protective immune responses that prevent severe CMV-associated fetal disease.

Cytomegalovirus (CMV) subunit vaccine candidates include glycoprotein B (gB), and phosphoprotein ppUL83 (pp65). Using a guinea pig cytomegalovirus (GPCMV) model, this study compared immunogenicity, pregnancy outcome, and congenital viral infection following pre-pregnancy immunization with a three-dose series of modified vaccinia virus Ankara (MVA)-vectored vaccines consisting either of gB administered alone, or simultaneously with a pp65 homolog (GP83)-expressing vaccine. Vaccinated and control dams were challenged at midgestation with salivary gland-adapted GPCMV. Comparisons included ELISA and neutralizing antibody responses, maternal viral load, pup mortality, and congenital infection rates. Strikingly, ELISA and neutralization titers were significantly lower in the gB/GP83 combined vaccine group than in the gB group. However, both vaccines protected against pup mortality (63.2% in controls vs. 11.4% and 13.9% in gB and gB/GP83 combination groups, respectively; p<0.0001). Reductions in pup viral load were noted for both vaccine groups compared to control, but preconception vaccination resulted in a significant reduction in GPCMV transmission only in the monovalent gB group (26/44, 59% v. 27/34, 79% in controls; p<0.05). We conclude that, using the MVA platform, the addition of GP83 to a gB subunit vaccine interferes with antibody responses and diminishes protection against congenital GPCMV infection, but does not decrease protection against pup mortality., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Human Cytomegalovirus (HCMV) utilizes two different pathways for host cell entry. HCMV entry into fibroblasts requires glycoproteins gB and gH/gL, whereas HCMV entry into epithelial and endothelial cells (EC) requires an additional complex composed of gH, gL, UL128, UL130, and UL131A, referred to as the gH/gL-pentamer complex (gH/gL-PC). While there are no established correlates of protection against HCMV, antibodies are thought to be important in controlling infection. Neutralizing antibodies (NAb) that prevent gH/gL-PC mediated entry into EC are candidates to be assessed for in vivo protective function. However, these potent NAb are predominantly directed against conformational epitopes derived from the assembled gH/gL-PC. To address these concerns, we constructed Modified Vaccinia Ankara (MVA) viruses co-expressing all five gH/gL-PC subunits (MVA-gH/gL-PC), subsets of gH/gL-PC subunits (gH/gL or UL128/UL130/UL131A), or the gB subunit from HCMV strain TB40/E. We provide evidence for cell surface expression and assembly of complexes expressing full-length gH or gB, or their secretion when the corresponding transmembrane domains are deleted. Mice or rhesus macaques (RM) were vaccinated three times with MVA recombinants and serum NAb titers that prevented 50% infection of human EC or fibroblasts by HCMV TB40/E were determined. NAb responses induced by MVA-gH/gL-PC blocked HCMV infection of EC with potencies that were two orders of magnitude greater than those induced by MVA expressing gH/gL, UL128-UL131A, or gB. In addition, MVA-gH/gL-PC induced NAb responses that were durable and efficacious to prevent HCMV infection of Hofbauer macrophages, a fetal-derived cell localized within the placenta. NAb were also detectable in saliva of vaccinated RM and reached serum peak levels comparable to NAb titers found in HCMV hyperimmune globulins. This vaccine based on a translational poxvirus platform co-delivers all five HCMV gH/gL-PC subunits to achieve robust humoral responses that neutralize HCMV infection of EC, placental macrophages and fibroblasts, properties of potential value in a prophylactic vaccine.

The guinea pig (Cavia porcellus) provides a useful animal model for studying the pathogenesis of many infectious diseases, and for preclinical evaluation of vaccines. However, guinea pig models are limited by the lack of immunological reagents required for characterization and quantification of antigen-specific T cell responses. To address this deficiency, an enzyme-linked immunospot (ELISPOT) assay for guinea pig interferon (IFN)-γ was developed to measure antigen/epitope-specific T cell responses to guinea pig cytomegalovirus (GPCMV) vaccines. Using splenocytes harvested from animals vaccinated with a modified vaccinia virus Ankara (MVA) vector encoding the GPCMV GP83 (homolog of human CMV pp65 [gpUL83]) protein, we were able to enumerate and map antigen-specific responses, both in vaccinated as well as GPCMV-infected animals, using a panel of GP83-specific peptides. Several potential immunodominant GP83-specific peptides were identified, including one epitope, LGIVHFFDN, that was noted in all guinea pigs that had a detectable CD8+ response to GP83. Development of a guinea pig IFN-γ ELISPOT should be useful in characterization of additional T cell-specific responses to GPCMV, as well as other pathogens. This information in turn can help focus future experimental evaluation of immunization strategies, both for GPCMV as well as for other vaccine-preventable illnesses studied in the guinea pig model., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Neutralizing antibodies (NAb) are important for interfering with horizontal transmission of human cytomegalovirus (HCMV) leading to primary and congenital HCMV infection. Recent findings have shown that a pentameric virion complex formed by the glycoproteins gH/gL, UL128, UL130, and UL131A (UL128C) is required for HCMV entry into epithelial/endothelial cells (Epi/EC) and is the target of potent NAb in HCMV-seropositive individuals. Using bacterial artificial chromosome technology, we have generated a modified vaccinia Ankara virus (MVA) that stably coexpresses all 5 rhesus CMV (RhCMV) proteins homologous to HCMV UL128C, termed MVA-RhUL128C. Coimmunoprecipitation confirmed the interaction of RhgH with the other 4 RhCMV subunits of the pentameric complex. All 8 RhCMV-naïve rhesus macaques (RM) vaccinated with MVA-RhUL128C developed NAb that blocked infection of monkey kidney epithelial cells (MKE) and rhesus fibroblasts. NAb titers induced by MVA-RhUL128C measured on both cell types at 2 to 6 weeks postvaccination were comparable to levels observed in naturally infected RM. In contrast, MVA expressing a subset of RhUL128C proteins or RhgB glycoprotein only minimally stimulated NAb that inhibited infection of MKE. In addition, following subcutaneous RhCMV challenge at 8 weeks postvaccination, animals vaccinated with MVA-RhUL128C showed reduced plasma viral loads. These results indicate that MVA expressing the RhUL128C induces NAb inhibiting RhCMV entry into both Epi/EC and fibroblasts and limits RhCMV replication in RM. This novel approach is the first step in developing a prophylactic HCMV vaccine designed to interfere with virus entry into major cell types permissive for viral replication, a required property of an effective vaccine.

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.

In order to facilitate the generation of mutant viruses of varicella-zoster virus (VZV), the agent causing varicella (chicken pox) and herpes zoster (shingles), we generated a full-length infectious bacterial artificial chromosome (BAC) clone of the P-Oka strain. First, mini-F sequences were inserted into a preexisting VZV cosmid, and the SuperCos replicon was removed. Subsequently, mini-F-containing recombinant virus was generated from overlapping cosmid clones, and full-length VZV DNA recovered from the recombinant virus was established in Escherichia coli as an infectious BAC. An inverted duplication of VZV genomic sequences within the mini-F replicon resulted in markerless excision of vector sequences upon virus reconstitution in eukaryotic cells. Using the novel tool, the role in VZV replication of the major tegument protein encoded by ORF9 was investigated. A markerless point mutation introduced in the start codon by two-step en passant Red mutagenesis abrogated ORF9 expression and resulted in a dramatic growth defect that was not observed in a revertant virus. The essential nature of ORF9 for VZV replication was ultimately confirmed by restoration of the growth of the ORF9-deficient mutant virus using trans-complementation via baculovirus-mediated gene transfer.