Stability of the HSV-2 US-6 Gene in the del II, del III, CP77, and I8R-G1L Sites in Modified Vaccinia Virus Ankara After Serial Passage of Recombinant Vectors in Cells

Atukorale, Vajini; Weir, Jerry P.; Meseda, Clement A.

doi:10.3390/vaccines8010137

Cited by 5 publications

(10 citation statements)

References 29 publications

(46 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Similarly, pUC57-S_ssT contained a synthetic SARS-CoV-2 sequence in which the sequence encoding the transmembrane domain and cytoplasmic tail was replaced with the sequence encoding a trimerization (foldon) sequence along with nucleotide substitutions that altered the S1/S2 protease cleavage site from residues RRAR to GSAS and residues 986/987 from KV to PP 6 . The two plasmids were used as templates for the amplification of sequences encoding spike protein forms (Table 1 ) for cloning into the MVA shuttle vector/Destination vector, pCAM18 19 . PCR amplicons were initially cloned into pENTR-D-TOPO vector (Invitrogen, Carlsbad, CA) to obtain pENTR-S, pENTR-ssM, and pENTR-RBD.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

MVA vector expression of SARS-CoV-2 spike protein and protection of adult Syrian hamsters against SARS-CoV-2 challenge

et al. 2021

Self Cite

View full text Add to dashboard Cite

Numerous vaccine candidates against SARS-CoV-2, the causative agent of the COVID-19 pandemic, are under development. The majority of vaccine candidates to date are designed to induce immune responses against the viral spike (S) protein, although different forms of S antigen have been incorporated. To evaluate the yield and immunogenicity of different forms of S, we constructed modified vaccinia virus Ankara (MVA) vectors expressing full-length S (MVA-S), the RBD, and soluble S ectodomain and tested their immunogenicity in dose-ranging studies in mice. All three MVA vectors induced spike-specific immunoglobulin G after one subcutaneous immunization and serum titers were boosted following a second immunization. The MVA-S and MVA-ssM elicited the strongest neutralizing antibody responses. In assessing protective efficacy, MVA-S-immunized adult Syrian hamsters were challenged with SARS-CoV-2 (USA/WA1/2020). MVA-S-vaccinated hamsters exhibited less severe manifestations of atypical pneumocyte hyperplasia, hemorrhage, vasculitis, and especially consolidation, compared to control animals. They also displayed significant reductions in gross pathology scores and weight loss, and a moderate reduction in virus shedding was observed post challenge in nasal washes. There was evidence of reduced viral replication by in situ hybridization, although the reduction in viral RNA levels in lungs and nasal turbinates did not reach significance. Taken together, the data indicate that immunization with two doses of an MVA vector expressing SARS-CoV-2 S provides protection against a stringent SARS-CoV-2 challenge of adult Syrian hamsters, reaffirm the utility of this animal model for evaluating candidate SARS-CoV-2 vaccines, and demonstrate the value of an MVA platform in facilitating vaccine development against SARS-CoV-2.

show abstract

Section: Methodsmentioning

confidence: 99%

“…MVA vectors were constructed as previously described 19 . Briefly, confluent monolayers of DF-1 cells in a 6-well tissue culture plate were infected with MVA at a multiplicity of infection (MOI) of 0.1 and transfected with 1 µg of a shuttle plasmid, using X-tremeGENE HP DNA transfection reagent (Sigma-Aldrich, St. Louis, MO).…”

Section: Methodsmentioning

confidence: 99%

MVA vector expression of SARS-CoV-2 spike protein and protection of adult Syrian hamsters against SARS-CoV-2 challenge

et al. 2021

Self Cite

View full text Add to dashboard Cite

show abstract

“…Instability of rMVA, i. e. the tendency to spontaneously loose transgene expression, is a common issue in the field (23)(24)(25)(26)(27). While generating rMVA vaccines against different viruses, e.g.…”

Section: Introductionmentioning

confidence: 99%

Transgene expression knock-down in recombinant Modified Vaccinia virus Ankara vectors improves genetic stability and sustained transgene maintenance across multiple passages

Neckermann,

Mohr,

Billmeier

et al. 2024

Front. Immunol.

View full text Add to dashboard Cite

Modified vaccinia virus Ankara is a versatile vaccine vector, well suited for transgene delivery, with an excellent safety profile. However, certain transgenes render recombinant MVA (rMVA) genetically unstable, leading to the accumulation of mutated rMVA with impaired transgene expression. This represents a major challenge for upscaling and manufacturing of rMVA vaccines. To prevent transgene-mediated negative selection, the continuous avian cell line AGE1.CR pIX (CR pIX) was modified to suppress transgene expression during rMVA generation and amplification. This was achieved by constitutively expressing a tetracycline repressor (TetR) together with a rat-derived shRNA in engineered CR pIX PRO suppressor cells targeting an operator element (tetO) and 3’ untranslated sequence motif on a chimeric poxviral promoter and the transgene mRNA, respectively. This cell line was instrumental in generating two rMVA (isolate CR19) expressing a Macaca fascicularis papillomavirus type 3 (MfPV3) E1E2E6E7 artificially-fused polyprotein following recombination-mediated integration of the coding sequences into the DelIII (CR19 M-DelIII) or TK locus (CR19 M-TK), respectively. Characterization of rMVA on parental CR pIX or engineered CR pIX PRO suppressor cells revealed enhanced replication kinetics, higher virus titers and a focus morphology equaling wild-type MVA, when transgene expression was suppressed. Serially passaging both rMVA ten times on parental CR pIX cells and tracking E1E2E6E7 expression by flow cytometry revealed a rapid loss of transgene product after only few passages. PCR analysis and next-generation sequencing demonstrated that rMVA accumulated mutations within the E1E2E6E7 open reading frame (CR19 M-TK) or deletions of the whole transgene cassette (CR19 M-DelIII). In contrast, CR pIX PRO suppressor cells preserved robust transgene expression for up to 10 passages, however, rMVAs were more stable when E1E2E6E7 was integrated into the TK as compared to the DelIII locus. In conclusion, sustained knock-down of transgene expression in CR pIX PRO suppressor cells facilitates the generation, propagation and large-scale manufacturing of rMVA with transgenes hampering viral replication.

show abstract

“…The MVA vector technology has demonstrated efficacy against HIV, influenza, parainfluenza, measles, flavivirus, and even malaria [ 30 ]. Atukorale et al developed a vaccinia virus vectored HSV-2 vaccine expressing gD2 [ 12 ]. However, the transgene was lost following the vaccine’s serial passage, pointing to potential vaccine stability issues.…”

Section: Introductionmentioning

confidence: 99%

Rational Design of Live-Attenuated Vaccines against Herpes Simplex Viruses

Stanfield

Kousoulas

Fernández³

et al. 2021

Viruses

View full text Add to dashboard Cite

Diseases caused by human herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) affect millions of people worldwide and range from fatal encephalitis in neonates and herpes keratitis to orofacial and genital herpes, among other manifestations. The viruses can be shed efficiently by asymptomatic carriers, causing increased rates of infection. Viral transmission occurs through direct contact of mucosal surfaces followed by initial replication of the incoming virus in skin tissues. Subsequently, the viruses infect sensory neurons in the trigeminal and lumbosacral dorsal root ganglia, where they are primarily maintained in a transcriptionally repressed state termed “latency”, which persists for the lifetime of the host. HSV DNA has also been detected in other sympathetic ganglia. Periodically, latent viruses can reactivate, causing ulcerative and often painful lesions primarily at the site of primary infection and proximal sites. In the United States, recurrent genital herpes alone accounts for more than a billion dollars in direct medical costs per year, while there are much higher costs associated with the socio-economic aspects of diseased patients, such as loss of productivity due to mental anguish. Currently, there are no effective FDA-approved vaccines for either prophylactic or therapeutic treatment of human herpes simplex infections, while several recent clinical trials have failed to achieve their endpoint goals. Historically, live-attenuated vaccines have successfully combated viral diseases, including polio, influenza, measles, and smallpox. Vaccines aimed to protect against the devastation of smallpox led to the most significant achievement in medical history: the eradication of human disease by vaccination. Recently, novel approaches toward developing safe and effective live-attenuated vaccines have demonstrated high efficacy in various preclinical models of herpetic disease. This next generation of live-attenuated vaccines has been tailored to minimize vaccine-associated side effects and promote effective and long-lasting immune responses. The ultimate goal is to prevent or reduce primary infections (prophylactic vaccines) or reduce the frequency and severity of disease associated with reactivation events (therapeutic vaccines). These vaccines’ “rational” design is based on our current understanding of the immunopathogenesis of herpesviral infections that guide the development of vaccines that generate robust and protective immune responses. This review covers recent advances in the development of herpes simplex vaccines and the current state of ongoing clinical trials in pursuit of an effective vaccine against herpes simplex virus infections and associated diseases.

show abstract

Stability of the HSV-2 US-6 Gene in the del II, del III, CP77, and I8R-G1L Sites in Modified Vaccinia Virus Ankara After Serial Passage of Recombinant Vectors in Cells

Cited by 5 publications

References 29 publications

MVA vector expression of SARS-CoV-2 spike protein and protection of adult Syrian hamsters against SARS-CoV-2 challenge

MVA vector expression of SARS-CoV-2 spike protein and protection of adult Syrian hamsters against SARS-CoV-2 challenge

Transgene expression knock-down in recombinant Modified Vaccinia virus Ankara vectors improves genetic stability and sustained transgene maintenance across multiple passages

Rational Design of Live-Attenuated Vaccines against Herpes Simplex Viruses

Contact Info

Product

Resources

About