Vaccinia viruses with mutations in the E3L gene as potential replication-competent, attenuated vaccines: Intra-nasal vaccination

Development of a vaccine to prevent congenital cytomegalovirus infection is a major public health priority. Live vaccines attenuated through mutations targeting viral mechanisms responsible for evasion of host defense may be both safe and efficacious. Safety and vaccine efficacy were evaluated using a guinea pig cytomegalovirus (GPCMV) model. Recombinant GPCMV with a targeted deletion of gp145 (designated ⌬145), a viral protein kinase R (PKR) inhibitor, was generated. Attenuation was evaluated following inoculation of 10 7 PFU of ⌬145 or parental virus into guinea pigs immunosuppressed with cyclophosphamide. Efficacy was evaluated by immunizing GPCMV-naive guinea pigs twice with either 10 5 or 10 6 PFU of ⌬145, establishing pregnancy, and challenging the guinea pigs with salivary gland-adapted GPCMV. The immune response, maternal viral load, pup mortality, and congenital infection rates in the vaccine and control groups were compared. ⌬145 was substantially attenuated for replication in immunocompromised guinea pigs. Vaccination with ⌬145 induced enzyme-linked immunosorbent assay (ELISA) and neutralizing antibody levels comparable to those achieved in natural infection. In the higher-and lower-dose vaccine groups, pup mortality was reduced to 1/24 (4%) and 4/29 (14%) pups, respectively, whereas it was 26/31 (81%) in unvaccinated control pups (P < 0.0001 for both groups versus the control group). Congenital infection occurred in 20/31 (65%) control pups but only 8/24 (33%) pups in the group vaccinated with 10 6 PFU (P < 0.05). Significant reductions in the magnitude of maternal DNAemia and pup viral load were noted in the vaccine groups compared to those in the controls. Deletion of a GPCMV genome-encoded PKR inhibitor results in a highly attenuated virus that is immunogenic and protective as a vaccine against transplacental infection. Infection with human cytomegalovirus (HCMV) causes considerable morbidity and occasional mortality in immunocompromised solid organ transplant and hematopoietic stem cell transplant patients, HIV-infected individuals, and newborns that acquire infection in utero (1, 2). Due to the lifelong morbidity associated with congenital CMV infection, a preconception vaccine capable of preventing virus transmission to the fetus would provide a highly cost-effective public health advance (3). Unfortunately, the lack of clear immunological correlates of protective immunity has hampered development of an HCMV vaccine. In spite of this uncertainty, there is evidence that virus-neutralizing antibody responses targeting viral envelope glycoproteins, as well as cellular immune responses (CD4 ϩ and CD8 ϩ ) targeting multiple structural and regulatory proteins, play important roles in protection against acquisition and reactivation of infection (4-7).

Section: Discussionmentioning

confidence: 99%

Vaccination with a Live Attenuated Cytomegalovirus Devoid of a Protein Kinase R Inhibitory Gene Results in Reduced Maternal Viremia and Improved Pregnancy Outcome in a Guinea Pig Congenital Infection Model

Schleiss

Bierle

Swanson

et al. 2015

“…model measuring weight loss; intracranial inoculations or measures of viral titers in organs have often been performed instead. Available data indicate that the A35⌬ virus is similar in virulence to the IL-18 binding protein knockout virus (36), more attenuated than the A46R deletion mutant (42), and less attenuated than the E3L deletion, which causes a 1,000-fold attenuation (46). The A35 gene affects the development of the host adaptive immune response.…”

Section: Discussionmentioning

confidence: 99%

The Poxvirus A35 Protein Is an Immunoregulator

Rehm

Jones

Tripp

et al. 2010

It was shown previously that the highly conserved vaccinia virus A35 gene is an important virulence factor in respiratory infection of mice. We show here that A35 is also required for full virulence by the intraperitoneal route of infection. A virus mutant in which the A35 gene has been removed replicated normally and elicited improved antibody, gamma interferon-secreting cell, and cytotoxic T-lymphocyte responses compared to wild-type virus, suggesting that A35 increases poxvirus virulence by immunomodulation. The enhanced immune response correlated with an improved control of viral titers in target organs after the development of the specific immune response. Finally, the A35 deletion mutant virus also provided protection from lethal challenge (1,000 50% lethal doses) equal to that of the wild-type virus. Together, these data suggest that A35 deletion viruses will make safer and more efficacious vaccines for poxviruses. In addition, the A35 deletion viruses will serve as improved platform vectors for other infectious diseases and cancer and will be superior vaccine choices for postexposure poxvirus vaccination, as they also provide improved kinetics of the immune response.

“…VACV mutants deleted of E3L (⌬E3L) or both K1L and C7L (⌬K1L⌬C7L) replicate abortively and express only a subset of viral genes in most mammalian cell lines (3, 24). These mutants are highly attenuated in animal hosts but are capable of eliciting immune responses, making them attractive vaccine vectors for infectious diseases and cancers (27,28). NYVAC, a VACV strain derived through deletion of 18 genes, including both K1L and C7L (27), has been used as the vector for an AIDS vaccine (2).…”

Section: Cellular Tropism Of Vaccinia Virus (Vacv) Ismentioning

confidence: 99%

Vaccinia Virus K1L and C7L Inhibit Antiviral Activities Induced by Type I Interferons

Meng

Jiang

Arsenio

et al. 2009

Cellular tropism of vaccinia virus (VACV) isVaccinia virus (VACV) is the prototypical member of the poxvirus family of large, complex, double-stranded DNA viruses (21). VACV has a very broad host range and is capable of infecting many vertebrate animal species. Its host range, however, can be significantly narrowed by deleting from its genome some of the so-called host range genes, the most important of which are E3L, K1L, and C7L (17). VACV mutants deleted of E3L (⌬E3L) or both K1L and C7L (⌬K1L⌬C7L) replicate abortively and express only a subset of viral genes in most mammalian cell lines (3, 24). These mutants are highly attenuated in animal hosts but are capable of eliciting immune responses, making them attractive vaccine vectors for infectious diseases and cancers (27,28). NYVAC, a VACV strain derived through deletion of 18 genes, including both K1L and C7L (27), has been used as the vector for an AIDS vaccine (2).The functions of E3L and the host factors that restrict the replication of the ⌬E3L mutant have been studied extensively. E3L encodes a 20-kDa and a 25-kDa protein that bind doublestranded RNA (dsRNA) and Z form DNA (6, 15). The E3 proteins antagonize the dsRNA-dependent protein kinase PKR (5), which exists as an inactive form in the cells and undergoes autophosphorylation and activation upon binding to dsRNA. The activated PKR phosphorylates the ␣ subunit of eukaryotic initiation factor 2 (eIF2␣), resulting in a block in protein translation at the initiation step. The infection of most mammalian cells by the ⌬E3L mutant leads to the activation of PKR and a block in translating viral mRNAs (16). The replication of the ⌬E3L mutant in nonpermissive HeLa cells can be rescued by silencing PKR expression (32), while its replication in permissive Huh7 (human hepatoma) cells can be blocked by upregulating PKR expression with interferon (IFN) treatment (1). In addition to affecting PKR, E3 has also been shown to inactivate IFN-stimulated gene 15 (ISG15) (14), another IFN effector that plays a role in host defense against VACV.Like the replication of the ⌬E3L mutant, the replication of the ⌬K1L⌬C7L mutant in nonpermissive HeLa cells is blocked at the translation of viral mRNA. However, the host factors that restrict the replication of the ⌬K1L⌬C7L mutant and the molecular functions of K1L or C7L remain a mystery. K1 and C7 share no amino acid sequence homology, but either K1L or C7L can complement the replication defect of the ⌬K1L⌬C7L mutant in most cell lines. The exception is rabbit RK13 cells, where K1L but not C7L can complement (24). K1L is present in only a few orthopoxviruses, while C7L or a functional homologue of C7L is present in almost all mammalian poxviruses (18). K1 comprises multiple ankyrin repeats, a protein motif that is involved in protein-ligand interaction. It was shown to prevent the degradation of IB␣ and to thus inhibit host . C7L has no homologue outside the poxvirus family, and its molecular function remains unknown. It may play a role in inhibiting cellular apoptosis in respons...