Jeronimo Cello

Mutation of a Single Conserved Nucleotide between the Cloverleaf and Internal Ribosome Entry Site Attenuates Poliovirus Neurovirulence

ABSTRACT The chemical synthesis of poliovirus (PV) cDNA combined with the cell-free synthesis of infectious particles yielded virus whose mouse neurovirulence was highly attenuated (J. Cello, A. V. Paul, and E. Wimmer, Science 297:1016-1018, 2002). Compared to the wild-type PV1 (Mahoney) [PV1(M)] sequence, the synthetic virus genome harbored 27 nucleotide (nt) changes deliberately introduced as genetic markers. Of the 27 nucleotide substitutions, the UA-to-GG exchanges at nucleotides 102/103, mapping to a region between the cloverleaf and the internal ribosome entry site (IRES) in the 5′-nontranslated region, were found to be involved in the observed attenuation phenotype in mice. The UA/GG mutation at nt 102/103 in the synthetic PV1(M) [sPV1(M)] background conferred also a ts phenotype of replication to the virus in human neuroblastoma cells. Conversely, the exchange of GG to wild-type (wt) UA at 102/103 in an sPV1(M) background restored wt neurovirulence in CD155 transgenic (tg) mice and suppressed the ts phenotype in SK-N-MC cells. All poliovirus variants replicated well in HeLa cells at the two temperatures, regardless of the sequence at the 102/103 locus. Analyses of variants isolated from sPV(M)-infected CD155 tg mice revealed that the G102G103-to-G102A103 reversion alone reestablished the neurovirulent phenotype. This suggests that a single mutation is responsible for the observed change of the neurovirulence phenotype. sPV1(M) RNA is translated in cell extracts of SK-N-MC cells with significantly lower efficiency than PV1(M) RNA or sPV1(M) RNA with a G102-to-A102 reversion. These studies suggest a function for the conserved nucleotide (A103) located between the cloverleaf and the IRES which is important for replication of PV in the central nervous system of CD155 tg mice and in human cells of neuronal origin.

A study of the cellular immune response to enteroviruses in humans: identification of cross-reactive T cell epitopes on the structural proteins of enteroviruses.

We have attempted to extend our understanding of the enteroviral cross-reactive T cell response in humans. Peripheral blood mononuclear cells (PBMC) from healthy donors were stimulated in vitro with six different serotypes of enterovirus and 15 synthetic peptides representing conserved regions in the four structural proteins of these viruses. Upon challenge with different antigens, PBMC from donors responded specifically with proliferation and production of interferon-gamma (IFN-gamma). In contrast, synthesis of interleukin-4 (IL-4) or IL-10 was not detected. A T cell response to each enterovirus serotype was recorded in all individuals even though not all individuals had serum neutralizing antibody against each virus. These data confirmed previous findings that human T cells recognize enteroviral cross-reactive epitopes. Analysis of the peptide-induced IFN-gamma production and proliferative response showed that the cross-reactive T cell epitopes are localized mainly in capsid protein VP2 and VP3 and to a lesser extent in VP1. Surprisingly, T cell epitopes were not identified in the most conserved structural protein of enterovirus, VP4. Immune responses were mediated by CD4+ T cells in association with MHC class II molecules. The sources of IFN-gamma in response to the most immunodominant cross-reactive T cell epitopes were CD4+, CD8+ and NK cells. The two latter subsets produced IFN-gamma provided CD4+ T cells were present. Since T helper 1 (Th1) cells can mediate an in vivo protective immune response against poliovirus infection in mice, our novel findings in humans merit further detailed characterization of T cells that recognize the enteroviral cross-reactive T cell epitopes.

Cold-Adapted Viral Attenuation (CAVA): Highly Temperature Sensitive Polioviruses as Novel Vaccine Strains for a Next Generation Inactivated Poliovirus Vaccine

The poliovirus vaccine field is moving towards novel vaccination strategies. Withdrawal of the Oral Poliovirus Vaccine and implementation of the conventional Inactivated Poliovirus Vaccine (cIPV) is imminent. Moreover, replacement of the virulent poliovirus strains currently used for cIPV with attenuated strains is preferred. We generated Cold-Adapted Viral Attenuation (CAVA) poliovirus strains by serial passage at low temperature and subsequent genetic engineering, which contain the capsid sequences of cIPV strains combined with a set of mutations identified during cold-adaptation. These viruses displayed a highly temperature sensitive phenotype with no signs of productive infection at 37°C as visualized by electron microscopy. Furthermore, decreases in infectious titers, viral RNA, and protein levels were measured during infection at 37°C, suggesting a block in the viral replication cycle at RNA replication, protein translation, or earlier. However, at 30°C, they could be propagated to high titers (9.4–9.9 Log10TCID50/ml) on the PER.C6 cell culture platform. We identified 14 mutations in the IRES and non-structural regions, which in combination induced the temperature sensitive phenotype, also when transferred to the genomes of other wild-type and attenuated polioviruses. The temperature sensitivity translated to complete absence of neurovirulence in CD155 transgenic mice. Attenuation was also confirmed after extended in vitro passage at small scale using conditions (MOI, cell density, temperature) anticipated for vaccine production. The inability of CAVA strains to replicate at 37°C makes reversion to a neurovirulent phenotype in vivo highly unlikely, therefore, these strains can be considered safe for the manufacture of IPV. The CAVA strains were immunogenic in the Wistar rat potency model for cIPV, inducing high neutralizing antibody titers in a dose-dependent manner in response to D-antigen doses used for cIPV. In combination with the highly productive PER.C6 cell culture platform, the stably attenuated CAVA strains may serve as an attractive low-cost and (bio)safe option for the production of a novel next generation IPV.

Brunenders: a partially attenuated historic poliovirus type I vaccine strain.

Brunenders, a type I poliovirus (PV) strain, was developed in 1952 by J. F. Enders and colleagues through serial in vitro passaging of the parental Brunhilde strain, and was reported to display partial neuroattenuation in monkeys. This phenotype of attenuation encouraged two vaccine manufacturers to adopt Brunenders as the type I component for their inactivated poliovirus vaccines (IPVs) in the 1950s, although today no licensed IPV vaccine contains Brunenders. Here we confirmed, in a transgenic mouse model, the report of Enders on the reduced neurovirulence of Brunenders. Although dramatically neuroattenuated relative to WT PV strains, Brunenders remains more virulent than the attenuated oral vaccine strain, Sabin 1. Importantly, the neuroattenuation of Brunenders does not affect in vitro growth kinetics and in vitro antigenicity, which were similar to those of Mahoney, the conventional type I IPV vaccine strain. We showed, by full nucleotide sequencing, that Brunhilde and Brunenders differ at 31 nucleotides, eight of which lead to amino acid changes, all located in the capsid. Upon exchanging the Brunenders capsid sequence with that of the Mahoney capsid, WT neurovirulence was regained in vivo, suggesting a role for the capsid mutations in Brunenders attenuation. To date, as polio eradication draws closer, the switch to using attenuated strains for IPV is actively being pursued. Brunenders preceded this novel strategy as a partially attenuated IPV strain, accompanied by decades of successful use in the field. Providing data on the attenuation of Brunenders may be of value in the further construction of attenuated PV strains to support the grand pursuit of the global eradication of poliomyelitis.

Production of high titer attenuated poliovirus strains on the serum-free PER.C6(®) cell culture platform for the generation of safe and affordable next generation IPV.

BACKGROUND As poliovirus eradication draws closer, alternative Inactivated Poliovirus Vaccines (IPV) are needed to overcome the risks associated with continued use of the Oral Poliovirus Vaccine and of neurovirulent strains used during manufacture of conventional (c) IPV. We have previously demonstrated the susceptibility of the PER.C6(®) cell line to cIPV strains; here we investigated the suspension cell culture platform for growth of attenuated poliovirus strains. METHODS We examined attenuated Sabin strain productivity on the PER.C6(®) cell platform compared to the conventional Vero cell platform. The suitability of the suspension cell platform for propagation of rationally-attenuated poliovirus strains (stabilized Sabin type 3 S19 derivatives and genetically attenuated and stabilized MonoCre(X) strains), was also assessed. Yields were quantified by infectious titer determination and D-antigen ELISA using either serotype-specific polyclonal rabbit sera for Sabin strains or monoclonal cIPV-strain-specific antibodies for cIPV, S19 and MonoCre(X) strains. RESULTS PER.C6(®) cells supported the replication of Sabin strains to yields of infectious titers that were in the range of cIPV strains at 32.5°C. Sabin strains achieved 30-fold higher yields (p<0.0001) on the PER.C6(®) cell platform as compared to the Vero cell platform in infectious titer and D-antigen content. Furthermore, Sabin strain productivity on the PER.C6(®) cell platform was maintained at 10l scale. Yields of infectious titers of S19 and MonoCre(X) strains were 0.5-1 log10 lower than seen for cIPV strains, whereas D-antigen yield and productivities in doses/ml using rationally-attenuated strains were in line with yields reported for cIPV strains. CONCLUSIONS Sabin and rationally-attenuated polioviruses can be grown to high infectious titers and D-antigen yields. Sabin strain infection shows increased productivity on the PER.C6(®) cell platform as compared to the conventional Vero cell platform. Novel cell platforms with the potential for higher yields could contribute to increased affordability of a next generation of IPV vaccines needed for achieving and maintaining poliovirus eradication.

Poliomyelitis in transgenic mice expressing CD155 under the control of the Tage4 promoter after oral and parenteral poliovirus inoculation.

An important step in poliovirus (PV) infection by the oral route in humans is replication of the virus in lymphatic tissues of the gastrointestinal (GI) tract, thought to be mainly in the Peyers patches of the small intestine. No immunocompetent transgenic (tg) mice that express human PV receptor (CD155) under the control of different promoters can be infected orally. The mouse orthologue of human CD155 is Tage4, a protein expressed at the surface of enterocytes and in the Peyers patches. We describe here the generation of a tg mouse model in which the Tage4 promoter was used to drive expression of the human PV receptor-coding region (Tage4-CD155tg mice). In this model, CD155 expression was observed by immunostaining in different regions in the Peyers patches but not in their germinal centres. Although a similar pattern of staining was observed between 3- and 6-week-old Tage4-CD155tg mice, poliomyelitis was only seen in the younger mice after PV infection by the oral route. When compared with TgPVR21 mice that expressed CD155 driven by its human promoter, 3-week-old Tage4-CD155tg mice were more susceptible to gut infection and paralysis following feeding with PV. Also, Tage4-CD155tg mice exhibited higher susceptibility to poliomyelitis after parenteral inoculation of PV. Remarkably, the LD50 after intracerebral inoculation of PV was similar in both CD155 tg mouse strains. The CD155 tg mouse model reported here, although moderately susceptible to oral infection, may be suitable to study mechanisms of PV replication in the gastrointestinal tract and to dissect important aspects of PV neuroinvasiveness.

Comparison of African, Asian, and American Zika Viruses in Swiss Webster mice: Virulence, neutralizing antibodies, and serotypes

The sequence of Zika virus has evolved as it has spread out of Africa and into the Americas. It is unclear whether American strains of the virus define a new serotype. Here, we have tested the virulence and immunogenicity of three wild-type ZIKV strains in neonatal Swiss Webster mice. We found that all three ZIKV strains (African MR766, 1947; Asian FSS13025, 2010; and American, PRVABC59, 2015) are capable of killing neonatal mice after intracranial injection. Intraperitoneal injection with these viruses did not kill, but produced neutralizing antibodies as measured by a PRNT50 assay. Sera from mice infected with each virus were tested for neutralizing activity against the infecting virus and also the other two viruses by a PRNT50 assay. In general, the antibodies induced by each virus were good at neutralizing that virus (the homologous virus), but somewhat poorer at neutralizing the other two viruses (heterologous viruses). Antibodies induced by the African strain MR766 were about 4-fold worse at neutralizing the American strain PRVABC59 than the homologous strain, while antibodies induced by the American strain were about 10-fold worse at neutralizing the African strain than the homologous strain. Because the antibodies are cross-neutralizing at some level, the viruses do not form separate serotypes. Nevertheless, these results raise concern that the immunity conferred by the African virus may protect only relatively poorly against the new American strains. This has implications for the possible spread of the American ZIKV strains to Africa and Southeast Asia, and also for the development of vaccines.

Eradicating polio: A balancing act.

The Editorial “eradicating polio” by A. Adams and D. M. Salisbury (6 November 2015, p. [609][1]) presented great news about the progress of polio eradication: Efforts led by the World Health Organization (WHO) resulted in only 70 cases of paralytic poliomyelitis caused by wild polioviruses of

Oncolytic Poliovirus Therapy in a Mouse Model of Neuroblastoma: Preclinical Data Analysis and Future Studies

Neuroblastoma, a malignant embryonal tumor of the neural crest cells, is one of the most common solid extracranial tumors of early childhood (Brodeur et al., 2006). The prevalence of neuroblastoma in children is 7.5 cases /100, 000 infants (Brodeur et al., 2006; Gao et al, 1997; Gurney et al., 1997; Spix et al., 2006). Furthermore, the annual incidence of this tumor is nearly 1.0/100,000 children under the age of 15 years (Ries et al., 2005). Neuroblastoma comprises about 8-10% of all childhood cancers and for approximately 15% of cancer deaths in children (Gao et al., 1997; Ries et al., 2005). Neuroblastoma can arise anywhere along the sympathetic nervous system. Fifty percent of the tumors originate in the adrenal medulla. Additional sites of origin include the nerve tissue in the chest, neck, pelvis or abdomen (Ries et al., 2005). Clinical presentation of neuroblastoma is highly variable and is dependent on the site of the primary tumor, as well as on the disease extent and on the absence or presence of paraneoplastic syndromes (Maris & Matthay, 1999; Park et al., 2008). The three main clinical scenarios are: localized, metastatic and 4S (S for special). In contrast to many other pediatric malignancies, progress in treatment of neuroblastoma (especially for advanced-stage tumors) has been relatively modest. Hence, at present, this tumor still poses a major challenge to the pediatric oncologist. Current treatment strategies include any or all of the following; watchfulwaiting, surgery, mild to aggressive chemotherapy, radiotherapy, and bone marrow transplants. Several new agents and combinations are in ongoing trials for relapsed neuroblastoma, including topoisomerase 1 inhibitors, radionuclides, histone deacetylase and tyrosine kinase inhibitors, monoclonal antibodies directed to disialoganglioside, and angiogenesis inhibitors (Maris & Matthay, 1999). As neuroblastoma is a disease that most often strikes young children, treating patients with aggressive therapy is a concern because of the potential for long-term health implications (from heart disease to second malignancies). Some children with neuroblastoma can be cured, and for these children, oncologists must try to give the minimum treatment possible while achieving cure. Although a fraction of the patients are cured with current treatments, approximately 40 per cent will die of this disease; for these patients improved treatment options are imperative (Matthay et al., 1999, 2009).