Expanding the Repertoire of Modified Vaccinia Ankara-Based Vaccine Vectors via Genetic Complementation Strategies

Abstract

Background Modified Vaccinia virus Ankara (MVA) is a safe, highly attenuated orthopoxvirus that is being developed as a recombinant vaccine vector for immunization against a number of infectious diseases and cancers. However, the expression by MVA vectors of large numbers of poxvirus antigens, which display immunodominance over vectored antigens-of-interest for the priming of T cell responses, and the induction of vector-neutralizing antibodies, which curtail the efficacy of subsequent booster immunizations, remain as significant impediments to the overall utility of such vaccines. Thus, genetic approaches that enable the derivation of MVA vectors that are antigenically less complex may allow for rational improvement of MVA-based vaccines.Principal FindingsWe have developed a genetic complementation system that enables the deletion of essential viral genes from the MVA genome, thereby allowing us to generate MVA vaccine vectors that are antigenically less complex. Using this system, we deleted the essential uracil-DNA-glycosylase (udg) gene from MVA and propagated this otherwise replication-defective variant on a complementing cell line that constitutively expresses the poxvirus udg gene and that was derived from a newly identified continuous cell line that is permissive for growth of wild type MVA. The resulting virus, MVAΔudg, does not replicate its DNA genome or express late viral gene products during infection of non-complementing cells in culture. As proof-of-concept for immunological ‘focusing’, we demonstrate that immunization of mice with MVAΔudg elicits CD8+ T cell responses that are directed against a restricted repertoire of vector antigens, as compared to immunization with parental MVA. Immunization of rhesus macaques with MVAΔudg-gag, a udg − recombinant virus that expresses an HIV subtype-B consensus gag transgene, elicited significantly higher frequencies of Gag-specific CD8 and CD4 T cells following both primary (2–4-fold) and booster (2-fold) immunizations as compared to the udg + control virus MVA-gag, as determined by intracellular cytokine assay. In contrast, levels of HIV Gag-specific antibodies were elicited similarly in macaques following immunization with MVAΔudg-gag and MVA-gag. Furthermore, both udg − and udg + MVA vectors induced comparatively similar titers of MVA-specific neutralizing antibody responses following immunization of mice (over a 4-log range: 104–108 PFU) and rhesus macaques. These results suggest that the generation of MVA-specific neutralizing antibody responses are largely driven by input MVA antigens, rather than those that are synthesized de novo during infection, and that the processes governing the generation of antiviral antibody responses are more readily saturated by viral antigen than are those that elicit CD8+ T cell responses.SignificanceOur identification of a spontaneously-immortalized (but not transformed) chicken embryo fibroblast cell line (DF-1) that is fully permissive for MVA growth and that can be engineered to stably express MVA genes provides the ba...

Deletion of Specific Immune-Modulatory Genes from Modified Vaccinia Virus Ankara-Based HIV Vaccines Engenders Improved Immunogenicity in Rhesus Macaques

“…We sought to determine whether MVA vectors with deletions of four different viral immune-modulatory genes, alone or in combination with a deletion of udg (30), elicit enhanced T cell responses against expressed HIV antigens (Gag and Env) compared to those elicited by a control vector that does not harbor these deletions and represents a parental (i.e., wildtype) MVA vector backbone. Toward this end, we constructed vaccine vectors that express identical synthetic, human codonoptimized, HIV subtype C consensus gag and env genes from early viral promoters at the MVA deletion III locus but that differ with regard to deletions of specific combinations of MVA immunemodulatory and DNA replication genes (MVA⌬4-HIV, MVA⌬5-HIV, and MVA-HIV [control]) ( Table 1; also see Table S1 in the supplemental material).…”

“…MVA⌬4-HIV harbors simultaneous deletions of four poxvirus immune-modulatory genes (MVA008L, MVA153L, MVA159R, and MVA184R), which encode an IL-18 binding protein (75,80), a CC-chemokine binding protein (7,62), a dominant negative Toll/IL-1 signaling adapter, and a soluble IL-1␤ receptor (3,76), respectively. MVA⌬5-HIV harbors a deletion of the viral uracil-DNA glycosylase gene (MVA101R) (30), which reduces late gene expression, in addition to the deletions of the above-described four viral immune-modulatory genes. Both modified vaccines and the nondeleted parental MVA vaccine express an identical set of synthetic, codon-optimized HIV genes encoding antigenic consensus subtype C Env and Gag proteins under the control of early poxvirus promoters (modified H5) from the MVA deletion III locus.…”

“…Additionally, the MVA⌬4-HIV vector harbors deletions of four viral immune evasion genes (MVA008L, MVA153L, MVA159R, and MVA184R), whereas the MVA⌬5-HIV vector harbors deletions of these four immune evasion genes in combination with the deletion of viral uracil-DNA glycosylase (MVA101R) (see Table S1 in the supplemental material). All vectors were generated and propagated and titers were determined by using a udg-complementing DF-1 fibroblastderived cell line, as previously described (30). Sucrose gradient-purified stocks of recombinant viruses were used for immunizations.…”

“…B5R-specific ELISAs utilized a recombinant B5R protein (BEI Resources) as the coating antigen and were performed analogously to those for HIV Env. Titers of MVAspecific binding and neutralizing antibodies were determined as described previously (30).…”

“…One approach toward improving the immunogenicity of MVA-based vaccines that we have pursued is based upon the deletion of an essential viral replication gene (udg) to abrogate viral late gene expression and thereby reduce the complexity of the repertoire of expressed vector antigens that may otherwise compete against antigens of interest (expressed from early viral promoters) for eliciting T and B cell responses (30). A complementary approach, which we report here, is to delete MVA immune-modulatory genes that are present in the vector genome and whose gene products may be predicted to interfere with the optimal generation of vaccine-induced cellular and humoral immunity.…”