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Modified H5 promoter improves stability of insert genes while maintaining immunogenicity during extended passage of genetically engineered MVA vaccines.

ABSTRACT: We have engineered recombinant (r) Modified Vaccinia Ankara (MVA) to express multiple antigens under the control of either of two related vaccinia synthetic promoters (pSyn) with early and late transcriptional activity or the modified H5 (mH5) promoter which has predominant early activity. We sequentially passaged these constructs and analyzed their genetic stability by qPCR, and concluded that rMVA expressing multiple antigens using the mH5 promoter exhibit remarkable genetic stability and maintain potent immunogenicity after serial passage. In contrast, rMVA expressing antigens using engineered vaccinia synthetic E/L (pSyn I or II) promoters are genetically unstable. Progressive accumulation of antigen loss variants resulted in a viral preparation with lower immunogenicity after serial passage. Metabolic labeling, followed by cold chase revealed little difference in stability of proteins expressed from mH5 or pSyn promoter constructs. We conclude that maintenance of genetic stability which is achieved using mH5, though not with pSyn promoters, is linked to timing, not the magnitude of expression levels of foreign antigen, which is more closely associated with immunogenicity of the vaccine.

SUBMITTER: Wang Z

PROVIDER: S-EPMC2821965 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Modified H5 promoter improves stability of insert genes while maintaining immunogenicity during extended passage of genetically engineered MVA vaccines.

Wang Zhongde Z Martinez Joy J Zhou Wendi W La Rosa Corinna C Srivastava Tumul T Dasgupta Anindya A Rawal Ravindra R Li Zhongqui Z Britt William J WJ Diamond Don D

Vaccine 20091205 6

We have engineered recombinant (r) Modified Vaccinia Ankara (MVA) to express multiple antigens under the control of either of two related vaccinia synthetic promoters (pSyn) with early and late transcriptional activity or the modified H5 (mH5) promoter which has predominant early activity. We sequentially passaged these constructs and analyzed their genetic stability by qPCR, and concluded that rMVA expressing multiple antigens using the mH5 promoter exhibit remarkable genetic stability and main ...[more]

PMID: 19969118

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Project description:BackgroundEradication of Plasmodium falciparum malaria will likely require a multivalent vaccine, but the development of a highly efficacious subunit-based formulation has been challenging. We previously showed that production and immunogenicity of two leading vaccine targets, PfMSP119 (blood-stage) and Pfs25 (sexual stage), could be enhanced upon genetic fusion to merozoite surface protein 8 (PfMSP8). Here, we sought to optimize a Pfs25-based formulation for use in combination with rPfMSP1/8 with the goal of maintaining the immunogenicity of each subunit.MethodsComparative mouse studies were conducted to assess the effects of adjuvant selection (Alhydrogel vs. glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE)) and antigen dose (2.5 vs. 0.5 μg) on the induction of anti-Pfs25 immune responses. The antibody response (magnitude, IgG subclass profile, and transmission-reducing activity (TRA)) and cellular responses (proliferation, cytokine production) generated in response to each formulation were assessed. Similarly, immunogenicity of a bivalent vaccine containing rPfMSP1/8 and rPfs25/8 was evaluated.ResultsAlum-based formulations elicited strong and comparable humoral and cellular responses regardless of antigen form (unfused rPfs25 or chimeric rPfs25/8) or dose. In contrast, GLA-SE based formulations elicited differential responses as a function of both parameters, with 2.5 μg of rPfs25/8 inducing the highest titers of functional anti-Pfs25 antibodies. Based on these data, chimeric rPfs25/8 was selected and tested in a bivalent formulation with rPfMSP1/8. Strong antibody titers against Pfs25 and PfMSP119 domains were induced with GLA-SE based formulations, with no indication of antigenic competition.ConclusionsWe were able to generate an immunogenic bivalent vaccine designed to target multiple parasite stages that could reduce both clinical disease and parasite transmission. The use of the same PfMSP8 carrier for two different vaccine components was effective in this bivalent formulation. As such, the incorporation of additional protective targets fused to the PfMSP8 carrier into the formulation should be feasible, further broadening the protective response.

Project description:The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates viral entry into cells expressing angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino-acid fragment of the 1,273-amino-acid S-protein protomer. The RBD is the primary SARS-CoV-2 neutralizing epitope and a critical target of any SARS-CoV-2 vaccine. Here, we show that this RBD conjugated to each of two carrier proteins elicited more potent neutralizing responses in immunized rodents than did a similarly conjugated proline-stabilized S-protein ectodomain. Nonetheless, the native RBD is expressed inefficiently, limiting its usefulness as a vaccine antigen. However, we show that an RBD engineered with four novel glycosylation sites (gRBD) is expressed markedly more efficiently and generates a more potent neutralizing responses as a DNA vaccine antigen than the wild-type RBD or the full-length S protein, especially when fused to multivalent carriers, such as a Helicobacter pylori ferritin 24-mer. Further, gRBD is more immunogenic than the wild-type RBD when administered as a subunit protein vaccine. Our data suggest that multivalent gRBD antigens can reduce costs and doses, and improve the immunogenicity, of all major classes of SARS-CoV-2 vaccines.IMPORTANCE All available vaccines for coronavirus disease 2019 (COVID-19) express or deliver the full-length SARS-CoV-2 spike (S) protein. We show that this antigen is not optimal, consistent with observations that the vast majority of the neutralizing response to the virus is focused on the S-protein receptor-binding domain (RBD). However, this RBD is not expressed well as an independent domain, especially when expressed as a fusion protein with a multivalent scaffold. We therefore engineered a more highly expressed form of the SARS-CoV-2 RBD by introducing four glycosylation sites into a face of the RBD normally occluded in the full S protein. We show that this engineered protein, gRBD, is more immunogenic than the wild-type RBD or the full-length S protein in both genetic and protein-delivered vaccines.

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Project description:The robustness of immune responses to an antigen could be dictated by the route of vaccine inoculation. Traditional smallpox vaccines, essentially vaccinia virus strains, that were used in the eradication of smallpox were administered by percutaneous inoculation (skin scarification). The modified vaccinia virus Ankara is licensed as a smallpox vaccine in Europe and Canada and currently undergoing clinical development in the United States. MVA is also being investigated as a vector for the delivery of heterologous genes for prophylactic or therapeutic immunization. Since MVA is replication-deficient, MVA and MVA-vectored vaccines are often inoculated through the intramuscular, intradermal or subcutaneous routes. Vaccine inoculation via the intramuscular, intradermal or subcutaneous routes requires the use of injection needles, and an estimated 10 to 20% of the population of the United States has needle phobia. Following an observation in our laboratory that a replication-deficient recombinant vaccinia virus derived from the New York City Board of Health strain elicited protective immune responses in a mouse model upon inoculation by tail scarification, we investigated whether MVA and MVA recombinants can elicit protective responses following percutaneous administration in mouse models. Our data suggest that MVA administered by percutaneous inoculation, elicited vaccinia-specific antibody responses, and protected mice from lethal vaccinia virus challenge, at levels comparable to or better than subcutaneous or intramuscular inoculation. High titers of specific neutralizing antibodies were elicited in mice inoculated with a recombinant MVA expressing the herpes simplex type 2 glycoprotein D after scarification. Similarly, a recombinant MVA expressing the hemagglutinin of attenuated influenza virus rgA/Viet Nam/1203/2004 (H5N1) elicited protective immune responses when administered at low doses by scarification. Taken together, our data suggest that MVA and MVA-vectored vaccines inoculated by scarification can elicit protective immune responses that are comparable to subcutaneous vaccination, and may allow for antigen sparing when vaccine supply is limited.

Project description:BackgroundSeveral SARS-CoV-2 vaccines have shown clinical efficacy against Covid-19 infection but there remains uncertainty about the immune responses elicited by different regimens. This is a particularly important question for older people who are at increased clinical risk following infection and in whom immune senescence may limit vaccine responses. The BNT162b2 mRNA and ChAdOx1 adenovirus vaccines were the first two vaccines deployed in the UK programme using an 8-12 week 'extended interval'.ObjectivesWe undertook analysis of the spike-specific antibody and cellular immune response in 131 participants aged 80+ years after the second dose of 'extended interval' dual vaccination with either BNT162b2 mRNA (n = 54) or ChAdOx1 (n = 77) adenovirus vaccine. Blood samples were taken 2-3 weeks after second vaccine and were paired with samples taken at 5-weeks after first vaccine which have been reported previously. Antibody responses were measured using the Elecsys® electrochemiluminescence immunoassay assay and cellular responses were assessed by IFN-γ ELISpot.ResultsAntibody responses against spike protein became detectable in all donors following dual vaccination with either vaccine. 4 donors had evidence of previous natural infection which is known to boost vaccine responses. Within the 53 infection-naïve donors the median antibody titre was 4030 U/ml (IQR 1892-8530) following BNT162b2 dual vaccination and 1405 (IQR 469.5-2543) in the 74 patients after the ChAdOx1 vaccine (p = < 0.0001). Spike-specific T cell responses were observed in 30% and 49% of mRNA and ChAdOx1 recipients respectively and median responses were 1.4-times higher in ChAdOx1 vaccinees at 14 vs 20 spots/million respectively (p = 0.022).ConclusionDual vaccination with BNT162b2 or ChAdOx1 induces strong humoral immunity in older people following an extended interval protocol. Antibody responses are 2.9-times higher following the mRNA regimen whilst cellular responses are 1.4-times higher with the adenovirus-based vaccine. Differential patterns of immunogenicity are therefore elicited from the two vaccine platforms. It will be of interest to assess the relative stability of immune responses after these homologous vaccine regimens in order to assess the potential need for vaccine boosting. Furthermore, these findings indicate that heterologous vaccine platforms may offer the opportunity to further optimize vaccine responses.

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Modified H5 promoter improves stability of insert genes while maintaining immunogenicity during extended passage of genetically engineered MVA vaccines.

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Modified H5 promoter improves stability of insert genes while maintaining immunogenicity during extended passage of genetically engineered MVA vaccines.

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