Pseudovirion Particle Production by Live Poxvirus Human Immunodeficiency Virus Vaccine Vector Enhances Humoral and Cellular Immune Responses

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Recombinant poxvirus vectors are undergoing intensive evaluation as vaccine candidates for a variety of infectious pathogens. Avipoxviruses, such as canarypox virus, are replication deficient in mammalian cells by virtue of a poorly understood species-specific restriction. Highly attenuated vaccinia virus strains such as modified vaccinia virus Ankara (MVA) are similarly unable to complete replication in most mammalian cells but have an abortive-late phenotype, in that the block to replication occurs post-virus-specific DNA replication. In this study, an identical expression cassette for human immunodeficiency virus gag, pro, and env coding sequences was placed in canarypox virus and MVA vector backbones in order to directly compare vector-borne expression and to analyze differences in vector-host cell interactions. Antigen production by recombinant MVA was shown to be greater than that from recombinant canarypox virus in the mammalian cell lines and in the primary human cells tested. This observation was primarily due to a longer duration of antigen production in recombinant MVA-infected cells. Apoptosis induction was found to be more profound with the empty canarypox virus vector than with MVA. Remarkably, however, the inclusion of a gag/pro/env expression cassette altered the kinetics of apoptosis induction in recombinant MVA-infected cells to levels equal to those found in canarypox virus-infected cells. Antigen production by MVA was noted to be greater in human dendritic cells and resulted in enhanced T-cell stimulation in an in vitro antigen presentation assay. These results reveal differences in poxvirus vector-host cell interactions that should be relevant to their use as immunization vehicles.

Section: Expression Of Hiv Proteins By Mva and Canarypox Virus Recombmentioning

confidence: 99%

Direct Comparison of Antigen Production and Induction of Apoptosis by Canarypox Virus- and Modified Vaccinia Virus Ankara-Human Immunodeficiency Virus Vaccine Vectors

Zhang

Cassis-Ghavami

Eller³

et al. 2007

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“…This idea has been attempted in the form of particle and cell-based vaccines (14,17,34,40,61,77). However, success in eliciting NAbs by these strategies has, to date, been limited, perhaps because the Env is not biochemically homogeneous.…”

mentioning

confidence: 99%

Enzyme Digests Eliminate Nonfunctional Env from HIV-1 Particle Surfaces, Leaving Native Env Trimers Intact and Viral Infectivity Unaffected

Crooks

Tong

Osawa

et al. 2011

138

HIV-1 viruses and virus-like particles (VLPs) bear nonnative "junk" forms of envelope (Env) glycoprotein that may undermine the development of antibody responses against functional gp120/gp41 trimers, thereby blunting the ability of particles to elicit neutralizing antibodies. Here, we sought to better understand the nature of junk Env with a view to devising strategies for its removal. Initial studies revealed that native trimers were surprisingly stable in the face of harsh conditions, suggesting that junk Env is unlikely to arise by trimer dissociation or gp120 shedding. Furthermore, the limited gp120 shedding that occurs immediately after synthesis of primary HIV-1 isolate Envs is not caused by aberrant cleavage at the tandem gp120/gp41 cleavage sites, which were found to cleave in a codependent manner. A major VLP contaminant was found to consist of an early, monomeric form of gp160 that is glycosylated in the endoplasmic reticulum (gp160ER) and then bypasses protein maturation and traffics directly into particles. gp160ER was found to bind two copies of monoclonal antibody (MAb) 2G12, consistent with its exclusively high-mannose glycan profile. These findings prompted us to evaluate enzyme digests as a way to remove aberrant Env. Remarkably, sequential glycosidase-protease digests led to a complete or nearcomplete removal of junk Env from many viral strains, leaving trimers and viral infectivity largely intact. "Trimer VLPs" may be useful neutralizing antibody immunogens.

“…A native context may naturally promote trimer stability without a need for modifications. However, progress in this area has been limited by the presence of nonfunctional Env in vaccine preparations (13,17,24,27,28,31,35,37,44,53,54,64). The antigenic "promiscuity" of nonfunctional Env appears to make it immunodominant in a vaccine setting, promoting overwhelmingly nonneutralizing responses at the expense of any neutralizing responses to native trimers (19).…”

mentioning

confidence: 99%

HIV-1 Virus-Like Particles Bearing Pure Env Trimers Expose Neutralizing Epitopes but Occlude Nonneutralizing Epitopes

Tong

Crooks

Osawa

et al. 2012

153

Hypothetically, since native HIV-1 Env trimers are exclusively recognized by neutralizing antibodies, they might induce the neutralizing antibodies in a vaccine setting. This idea has not been evaluated due to the difficulty of separating trimers from nonfunctional Env (uncleaved gp160 and gp41 stumps). The latter are immunodominant and induce nonneutralizing antibodies. We previously showed that nonfunctional Env can be selectively cleared from virus-like particle (VLP) surfaces by enzyme digests (E. T. Crooks, T. Tong , K. Osawa, and J. M. Binley, J.Virol. 85:5825, 2011). Here, we investigated the effects of these digests on the antigenicity of VLPs and their sensitivity to neutralization. Before digestion, WT VLPs (bearing wild-type Env) and UNC VLPs (bearing uncleaved gp160) were recognized by various Env-specific monoclonal antibodies (MAbs), irrespective of their neutralizing activity, a result which is consistent with the presence of nonfunctional Env. After digestion, only neutralizing MAbs recognized WT VLPs, consistent with selective removal of nonfunctional Env (i.e., "trimer VLPs"). Digests eliminated the binding of all MAbs to UNC VLPs, again consistent with removal of nonfunctional Env. An exception was MAb 2F5, which weakly bound to digested UNC VLPs and bald VLPs (bearing no Env), perhaps due to lipid cross-reactivity. Trimer VLPs were infectious, and their neutralization sensitivity was largely comparable to that of undigested WT VLPs. However, they were ϳ100-fold more sensitive to the MAbs 4E10 and Z13e1, suggesting increased exposure of the gp41 base. Importantly, a scatterplot analysis revealed a strong correlation between MAb binding and neutralization of trimer VLPs. This suggests that trimer VLPs bear essentially pure native trimer that should allow its unfettered evaluation in a vaccine setting. Broadly neutralizing antibodies (bnAbs) are widely expected to be a crucial component of the immunity imparted by an effective HIV-1 vaccine (32, 42). These bnAbs neutralize the virus by being able to bind to native trimeric Envelope glycoprotein (Env) spikes on HIV-1 particle surfaces, thereby interfering with receptor engagement and infection (16,26,30,52). These Env spikes consist of trimers of gp120/gp41 heterodimers, in which gp120 is the surface subunit and gp41 is the transmembrane-anchoring subunit. By virtue of their compact and highly glycosylated nature, Env spikes effectively resist binding by all but a few rare neutralizing monoclonal antibodies (MAbs).To date, most Env-based vaccine candidates induce antibody responses against determinants that are inaccessible on native Env spikes and have therefore failed to induce meaningful neutralizing Ab responses (65). As the natural target of neutralizing Abs, the authentic Env spike in a native membrane context might fare better as an immunogen: logically, any antibodies induced by native Env trimers in a vaccine setting might be expected to neutralize. Most work in this area has centered on generating soluble Env trimers. However, the produ...