Second-Generation Rabies Virus-Based Vaccine Vectors Expressing Human Immunodeficiency Virus Type 1 Gag Have Greatly Reduced Pathogenicity but Are Highly Immunogenic

Key problems using viral vectors for vaccination and gene therapy are antivector immunity, low transduction efficiencies, acute toxicity, and limited capacity to package foreign genetic information. It could be demonstrated that animal and human cells were efficiently transduced with equine herpesvirus 1 (EHV-1) reconstituted from viral DNA maintained and manipulated in Escherichia coli. Between 13 and 23% of primary human CD3 ؉ , CD4 ؉ , CD8 ؉ , CD11b ؉ , and CD19 ؉ cells and more than 70% of CD4 ؉ MT4 cells or various human tumor cell lines (MeWo, Huh7, HeLa, 293T, or H1299) could be transduced with one infectious unit of EHV-1 per cell. After intranasal instillation of EHV-1 into mice, efficient transgene expression in lungs was detectable. Successful immunization using EHV-1 was shown after delivery of the human immunodeficiency virus type 1 Pr55 gag precursor by the induction of a Gag-specific CD8 ؉ immune response in mice. Because EHV-1 was not neutralized by human sera containing high titers of antibodies directed against human herpesviruses 1 to 5, it is concluded that this animal herpesvirus has enormous potential as a vaccine vector, because it is able to efficiently transduce a variety of animal and human cells, has high DNA packaging capacity, and can conveniently be maintained and manipulated in prokaryotic cells.Equine herpesvirus 1 (EHV-1), an alphaherpesvirus, causes infections in equines. Usually the disease induced by the virus is mild, and approximately 90% of equines worldwide harbor the agent, whose persistence in animals is lifelong. One of the peculiarities of EHV-1 is its tropism for blood mononuclear cells, which are the primary site for establishment of EHV-1 latency. Neuronal tissues, including the trigeminal ganglion and the brain, are only rarely infected by the agent. Neurological symptoms after infection of equines with certain EHV-1 strains are caused by infection of endothelial cells and subsequent hypoxia and neuronal degradation and are consistent with myeloencephalopathy and not myeloencephalitis (40,62).Herpesviridae enter target cells by fusion of the viral envelope with the plasma membrane at neutral pH after attachment of virions to cell surface glycosaminoglycans (47,51). Glycoproteins are crucially involved in these early stages of infection, and 11 glycoproteins in the prototype member of the Alphaherpesvirinae subfamily, Herpes simplex virus type 1 (HSV-1), have been identified. The herpesvirus glycoproteins involved in attachment, receptor binding, and fusion (i.e., gB, gC, gD, and the gH-gL complex) also appear to largely determine the mostly very restricted host tropism of Alphaherpesvirinae, i.e., the inability of animal viruses to naturally infect species other than those they have coevolved with (23). While the first contact and heparin-sensitive attachment of the studied alphaherpesviruses, including EHV-1, is mainly mediated by gC, receptor binding of HSV-1, pseudorabies virus, and bovine herpesvirus 1 (BHV-1) is via interaction of gD with cellular receptors th...

Section: Discussionmentioning

confidence: 99%

Potential of Equine Herpesvirus 1 as a Vector for Immunization

Trapp

Einem

Hofmann

et al. 2005

“…SPBN, a RABV vaccine strain-based vector, has been described previously (27). Vesicular stomatitis virus expressing green fluorescent protein (VSV-GFP) was obtained from John Hiscott (McGill University) (17).…”

Section: Methodsmentioning

confidence: 99%

Rabies Virus Is Recognized by the NLRP3 Inflammasome and Activates Interleukin-1β Release in Murine Dendritic Cells

Lawrence

Hudacek

Zoete

et al. 2013

Self Cite

Inflammasome activation is important for the development of an effective host defense against many pathogens, including RNA viruses. However, the mechanism by which the inflammasome recognizes RNA viruses and its role in rabies virus (RABV) pathogenicity and immunogenicity remain poorly defined. To determine the function of the inflammasome in response to RABV infection, we infected murine bone marrow-derived dendritic cells (BMDCs) with RABV. Our results indicate that the infection of BMDCs with RABV induces both the production of pro-interleukin-1␤ (pro-IL-1␤) and its processing, resulting in the secretion of active IL-1␤ through activation of the NLRP3-, ASC-, and caspase-1-dependent inflammasome. As previously shown for the induction of type I interferon by RABV, the induction of pro-IL-1␤ also depends upon IPS-1. We demonstrate that both the production of pro-IL-1␤ and activation of the inflammasome require viral replication. We also demonstrate that increased viral replication in BMDCs derived from IFNAR-deficient mice resulted in significantly more IL-1␤ release. Additionally, IL-1 receptor-deficient mice show an increase in RABV pathogenicity. Taken together, these results indicate an important role of the inflammasome in innate immune recognition of RABV.

“…The final plasmid was designated pCAGGs-4A. To construct the recombinant RV containing the respective M mutations the plasmids pCAGGs-35S, pCAGGs-36A, pCAGGs-38A, pCAGGs-41A, and pCAGGs-4A were digested with BstZ17I and SnaBI and the resulting fragment cloned into pSPBN (33). The resulting plasmids were designated pM35S, pM36A, pM38A, pM41A, and pM4A.…”

Section: Methodsmentioning

confidence: 99%

PPEY Motif within the Rabies Virus (RV) Matrix Protein Is Essential for Efficient Virion Release and RV Pathogenicity

Wirblich

Tan²,

Papaneri

et al. 2008

Self Cite

Late (L) domains containing the highly conserved sequence PPXY were first described for retroviruses, and later research confirmed their conservation and importance for efficient budding of several negative-stranded RNA viruses. Rabies virus (RV), a member of the Rhabdoviridae family, contains the sequence PPEY (amino acids 35 to 38) within the N terminus of the matrix (M) protein, but the functions of this potential L-domain in the viral life cycle, viral pathogenicity, and immunogenicity have not been established. Here we constructed a series of recombinant RVs containing mutations within the PPEY motif and analyzed their effects on viral replication and RV pathogenicity. Our results indicate that the first proline at position 35 is the most important for viral replication, whereas P36 and Y38 have a lesser but still noticeable impact. The reduction in viral replication was most likely due to inhibition of virion release, because initially no major impact on RV RNA synthesis was observed. In addition, results from electron microscopy demonstrated that the M4A mutant virus (PPEY3SAEA) displayed a more cell-associated phenotype than that of wild-type RV. Furthermore, all mutations within the PPEY motif resulted in reduced spread of the recombinant RVs as indicated by a reduction in focus size. Importantly, recombinant PPEY L-domain mutants were highly attenuated in mice yet still elicited potent antibody responses against RV G protein that were as high as those observed after infection with wild-type virus. Our data indicate that the RV PPEY motif has L-domain activity essential for efficient virus production and pathogenicity but is not essential for immunogenicity and thus can be targeted to increase the safety of rabies vaccine vectors.Rabies virus (RV) is a nonsegmented negative-stranded RNA of the genus Lyssavirus within the Rhabdoviridae family (19). Rhabdoviruses, such as RV and vesicular stomatitis virus (VSV), are simple enveloped RNA viruses which encode only five proteins: a nucleoprotein (N), which encapsidates the genomic and antigenomic RNA and forms the ribonucleoprotein (RNP); a phosphoprotein (P), which is the noncatalytic subunit of the RNA polymerase complex; a matrix protein (M); a transmembrane glycoprotein (G); and the viral polymerase (L) (54). Whereas the RNP is the template for viral transcription and replication (4), the RV G and M are the key players in viral budding and assembly (38-40, 43, 46). In the case of RV G, it has been shown that this protein is not essential for viral budding, but the amount of virions is reduced about 30-fold if RV G is deleted (39). In addition, Mebatsion et al. showed that the RV G cytoplasmic domain (CD) specifically interacts with the RV M protein and that a CD-deleted glycoprotein reduces RV budding about sixfold (39,40). This is in contrast to VSV, for which it has been shown that only a CD is required and not a specific amino acid sequence (46). However, similar to RV, VSV can also bud without its own glycoprotein but with also a reduced efficiency. ...