Recombinant proteins produced by vaccinia virus vectors can be incorporated within the virion (IMV form) into different compartments

Abstract

Vaccinia virus (VV) is one of the largest and most complex of animal viruses, with a virion that contains about 100 different polypeptides. Assembly of the viral proteins occurs in discrete cytoplasmic sites leading to formation of two infectious forms, an abundant (>90%) intracellular mature virus (IMV) with an envelope, and a minor extracellular enveloped virus (EEV) with an extra membrane acquired from the trans-Golgi network. It has been shown that while EEV contains in the outer membrane cellular proteins probably acquired during virus release from cells, however, IMV exclude host proteins during assembly. Since VV recombinants (VVr) expressing genes of interest are candidates as potential vaccines against pathogens and cancer, it becomes important to know if VVr can acquire foreign proteins during morphogenesis. In this investigation we show that purified virions (IMVs) from VVr can incorporate foreign proteins into different sites in the virus particle. By sequential fractionation of virion compartments with detergents, we found foreign proteins in the lipid envelope (cytokine IL-12 and CS antigen of Plasmodium yoelii), as part of a protein matrix-like membrane (HIV-1 gp41 of env), or more closely associated with the core containing the DNA complexes (HIV-1 gp160; a multiepitopic protein with the V3 loop of HIV-1 Env from different isolates, and beta-galactosidase). Similar findings were observed with purified virions derived from the WR strain as well as from the highly attenuated modified vaccinia virus Ankara (MVA) strain. These observations should be taken into consideration when VVr are used in clinical trials or in other vaccination approaches.

Fine structure of the vaccinia virion determined by controlled degradation and immunolocalization

“…Interestingly, neither G5 nor G6 have been identified in any of the published proteomic studies. Because in these studies G5 and G6 were expressed under a T7 operator it is possible that under these conditions both proteins were overexpressed and passively encapsidated by the virions (Franke and Hruby, 1987; Gomez and Esteban, 2001). We showed that the treatment of cores with higher a concentration of 100 mM DTT and 3M NaCl could remove proteins from the surface of the core while preserving its structure.…”

Attenuation of vaccinia virus by the expression of human Flt3 ligand

“…Vaccinia virus expressing the bacterial CAT gene incorporated the enzyme into the virus particle [46]. The expression of the cytokine IL-12 led to the incorporation of about 0.01% of the total recombinant protein into an envelope fraction, HIV1 env (gp160-120) was tightly bound in protein-DNA complexes, and the enzyme beta-galactosidase was found exclusively in core-associated fraction [47]. Transport and sorting of viral proteins directly from the endoplasmic reticulum into the growing immature virions using non-COPII vesicles [48] could explain selective integration of the low-glycosylated FL into P13-E/L-FL virions.…”

Immune properties of recombinant vaccinia virus encoding CD154 (CD40L) are determined by expression of virally encoded CD40L and the presence of CD40L protein in viral particles

“…This finding is not unexpected, particularly with recent evidence suggesting that intracellular expression of proteins from rVV transgenes often leads to incorporation of these proteins in both EEV and the more prevalent intracellular mature virion (IMV) particles. 25,26 Moreover, it has been shown that proteins originating from host cells and carried by the virus might be biologically active as exemplified by retroviral vectors produced in FasL-expressing cell lines. 39 The differences in IL-12 synthesis in DC exposed to replicating or nonreplicating rV-CD40L, or to MC38 cells infected with either of the viruses, indicate that the biologic effect of rV-CD40L is difficult to predict and likely depends on the diverse effects of CD40L protein carried by the viral particles, intracellular expression of CD40L and the adjuvant properties as well as cytopathic effects of the viral vector.…”

“…23,24 Viral immunogenicity may also be influenced by cellular antigens that are carried by VV particles acquired from host cells in culture. 25,26 This occurs due to the life cycle of VV within the cytoplasm of infected cells, where intracellular membranes are incorporated into intracellular enveloped virus (IEV) or cell membrane components are captured by the virus during assembly of extracellular enveloped virus (EEV) particles. The immunologic consequences of these ''captured proteins'' have not been fully elucidated in vaccination models, but could theoretically influence the quality of the immune response.…”