Viruses that replicate in the cytoplasm cannot access the host nuclear capping machinery. These viruses have evolved viral methyltransferase(s) to methylate N-7 and 2=-O cap of their RNA; alternatively, they "snatch" host mRNA cap to form the 5= end of viral RNA. The function of 2=-O methylation of viral RNA cap is to mimic cellular mRNA and to evade host innate immune restriction. A cytoplasmic virus defective in 2=-O methylation is replicative, but its viral RNA lacks 2=-O methylation and is recognized and eliminated by the host immune response. Such a mutant virus could be rationally designed as a live attenuated vaccine. Here, we use Japanese encephalitis virus (JEV), an important mosquito-borne flavivirus, to prove this novel vaccine concept. We show that JEV methyltransferase is responsible for both N-7 and 2=-O cap methylations as well as evasion of host innate immune response. Recombinant virus completely defective in 2=-O methylation was stable in cell culture after being passaged for >30 days. The mutant virus was attenuated in mice, elicited robust humoral and cellular immune responses, and retained the engineered mutation in vivo. A single dose of immunization induced full protection against lethal challenge with JEV strains in mice. Mechanistically, the attenuation phenotype was attributed to the enhanced sensitivity of the mutant virus to the antiviral effects of interferon and IFIT proteins. Collectively, the results demonstrate the feasibility of using 2=-O methylationdefective virus as a vaccine approach; this vaccine approach should be applicable to other flaviviruses and nonflaviviruses that encode their own viral 2=-O methyltransferases. Live attenuated vaccine represents the best medical intervention to prevent many viral diseases, such as those caused by vaccinia virus, poliovirus (Sabin), yellow fever virus (YFV; YF-17D), Japanese encephalitis virus (JEV SA14-14-2), and MMR (measles, mumps, and rubella viruses). The attenuated vaccine replicates to a low level but induces immune response and memory that are sufficient to prevent virulent virus infection. The traditional method of developing an attenuated vaccine is by passaging the virus through a foreign host (e.g., tissue culture or live animals). The attenuation of a vaccine strain is empirically achieved through accumulation of random mutations during passaging while maintaining immunogenicity. The function of each accumulated mutation in the vaccine strain needs to be analyzed to understand the mechanism of attenuation. As an alternative approach for vaccine development, viral attenuation could be rationally designed by altering the ability of virus to antagonize innate immunity (1). Such rationally designed virus is replicative and induces protective immunity; however, the virus is quickly eliminated due to its enhanced sensitivity to the antiviral effect of the host innate immune response.RNA and DNA viruses that replicate in the cytoplasm cannot use the cellular nuclear capping machinery and thus have evolved viral methyltransferas...

A therapy for dengue is still elusive. We describe the neutralizing and protective capacity of a dengue serotype-cross-reactive antibody isolated from the plasmablasts of a patient. Antibody SIgN-3C neutralized all four dengue virus serotypes at nano to picomolar concentrations and significantly decreased viremia of all serotypes in adult mice when given 2 days after infection. Moreover, mice were protected from pathology and death from a lethal dengue virus-2 infection. To avoid potential Fc-mediated uptake of immune complexes and ensuing enhanced infection, we introduced a LALA mutation in the Fc part. SIgN-3C-LALA was as efficient as the non-modified antibody in neutralizing dengue virus and in protecting mice while antibody-dependent enhancement was completely abrogated. The epitope of the antibody includes conserved amino acids in all three domains of the glycoprotein, which can explain its cross-reactivity. SIgN-3C-LALA neutralizes dengue virus both pre and post-attachment to host cells. These attributes likely contribute to the remarkable protective capacity of SIgN-3C.

Dengue is endemic in tropical countries worldwide and the four dengue virus serotypes often co-circulate. Infection with one serotype results in high titers of cross-reactive antibodies produced by plasmablasts, protecting temporarily against all serotypes, but impairing protective immunity in subsequent infections. To understand the development of these plasmablasts, we analyzed virus-specific B cell properties in patients during acute disease and at convalescence. Plasmablasts were unrelated to classical memory cells expanding in the blood during early recovery. We propose that only a small subset of memory B cells is activated as plasmablasts during repeat infection and that plasmablast responses are not representative of the memory B cell repertoire after dengue infection.

Dengue virus has four serotypes and is endemic globally in tropical countries. Neither a specific treatment nor an approved vaccine is available, and correlates of protection are not established. The standard neutralization assay cannot differentiate between serotype-specific and serotype cross-reactive antibodies in patients early after infection, leading to an overestimation of the long-term serotype-specific protection of an antibody response. It is known that the cross-reactive response in patients is temporary but few studies have assessed kinetics and potential changes in serum antibody specificity over time. To better define the specificity of polyclonal antibodies during disease and after recovery, longitudinal samples from patients with primary or secondary DENV-2 infection were collected over a period of 1 year. We found that serotype cross-reactive antibodies peaked 3 weeks after infection and subsided within 1 year. Since secondary patients rapidly produced antibodies specific for the virus envelope (E) protein, an E-specific ELISA was superior compared to a virus particle-specific ELISA to identify patients with secondary infections. Dengue infection triggered a massive activation and mobilization of both naïve and memory B cells possibly from lymphoid organs into the blood, providing an explanation for the surge of circulating plasmablasts and the increase in cross-reactive E protein-specific antibodies.

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The original version of the Article contained errors in the display of the labels in Fig. 6b. These errors have now been corrected in the HTML and PDF versions of the paper.