RESUMEN

Dengue virus (DENV) is responsible for the most prevalent and significant arthropod-borne viral infection of humans. The leading DENV vaccines are based on tetravalent live-attenuated virus platforms. In practice, it has been challenging to induce balanced and effective responses to each of the four DENV serotypes because of differences in the replication efficiency and immunogenicity of individual vaccine components. Unlike live vaccines, tetravalent DENV envelope (E) protein subunit vaccines are likely to stimulate balanced immune responses, because immunogenicity is replication independent. However, E protein subunit vaccines have historically performed poorly, in part because the antigens utilized were mainly monomers that did not display quaternary-structure epitopes found on E dimers and higher-order structures that form the viral envelope. In this study, we compared the immunogenicity of DENV2 E homodimers and DENV2 E monomers. The stabilized DENV2 homodimers, but not monomers, were efficiently recognized by virus-specific and flavivirus cross-reactive potently neutralizing antibodies that have been mapped to quaternary-structure epitopes displayed on the viral surface. In mice, the dimers stimulated 3-fold-higher levels of virus-specific neutralizing IgG that recognized epitopes different from those recognized by lower-level neutralizing antibodies induced by monomers. The dimer induced a stronger E domain I (EDI)- and EDII-targeted response, while the monomer antigens stimulated an EDIII epitope response and induced fusion loop epitope antibodies that are known to facilitate antibody-dependent enhancement (ADE). This study shows that DENV E subunit antigens that have been designed to mimic the structural organization of the viral surface are better vaccine antigens than E protein monomers.IMPORTANCE Dengue virus vaccine development is particularly challenging because vaccines have to provide protection against four different dengue virus stereotypes. The leading dengue virus vaccine candidates in clinical testing are all based on live-virus vaccine platforms and struggle to induce balanced immunity. Envelope subunit antigens have the potential to overcome these limitations but have historically performed poorly as vaccine antigens, because the versions tested previously were presented as monomers and not in their natural dimer configuration. This study shows that the authentic presentation of DENV2 E-based subunits has a strong impact on antibody responses, underscoring the importance of mimicking the complex protein structures that are found on DENV particle surfaces when designing subunit vaccines.

Asunto(s)

Anticuerpos Monoclonales/biosíntesis , Anticuerpos Antivirales/biosíntesis , Vacunas contra el Dengue/administración & dosificación , Dengue/prevención & control , Epítopos/inmunología , Vacunación/métodos , Proteínas del Envoltorio Viral/inmunología , Animales , Acrecentamiento Dependiente de Anticuerpo , Chlorocebus aethiops , Reacciones Cruzadas , Dengue/inmunología , Dengue/patología , Dengue/virología , Vacunas contra el Dengue/genética , Vacunas contra el Dengue/inmunología , Virus del Dengue/efectos de los fármacos , Virus del Dengue/genética , Virus del Dengue/inmunología , Modelos Animales de Enfermedad , Epítopos/química , Epítopos/genética , Femenino , Células HEK293 , Humanos , Inmunogenicidad Vacunal , Ratones , Ratones Endogámicos BALB C , Isoformas de Proteínas/administración & dosificación , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Multimerización de Proteína/efectos de los fármacos , Vacunas de Subunidad , Células Vero , Proteínas del Envoltorio Viral/administración & dosificación , Proteínas del Envoltorio Viral/genética

RESUMEN

The current leading Zika vaccine candidates in clinical testing are based on live or killed virus platforms, which have safety issues, especially in pregnant women. Zika subunit vaccines, however, have shown poor performance in preclinical studies, most likely because the antigens tested do not display critical quaternary structure epitopes present on Zika E protein homodimers that cover the surface of the virus. Here, we produce stable recombinant E protein homodimers that are recognized by strongly neutralizing Zika specific monoclonal antibodies. In mice, the dimeric antigen stimulate strongly neutralizing antibodies that target epitopes that are similar to epitopes recognized by human antibodies following natural Zika virus infection. The monomer antigen stimulates low levels of E-domain III targeting neutralizing antibodies. In a Zika challenge model, only E dimer antigen stimulates protective antibodies, not the monomer. These results highlight the importance of mimicking the highly structured flavivirus surface when designing subunit vaccines.

Asunto(s)

Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/metabolismo , Vacunas Virales/inmunología , Virus Zika/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Chlorocebus aethiops , Epítopos/inmunología , Femenino , Humanos , Ratones Endogámicos C57BL , Multimerización de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Células Vero , Proteínas del Envoltorio Viral/genética , Virus Zika/genética , Infección por el Virus Zika/inmunología , Infección por el Virus Zika/virología

RESUMEN

The epithelial content of an estradiol-sensitive immunological marker (CVA) has been quantified by mixed haemagglutination on tissue sections from the vagina of neonatal mice exposed to different schedules of estradiol treatment. Daily administration of estradiol-17ß (5 µg/day) was especially efficient in elevating the CVA content when the hormone was administered during the first four days after birth. Following a single injection of estradiol-17ß (5 µg in aqueous suspension) on one of the first five days of life, the vaginal epithelium reacted with a more vigourous CVA accumulation when challenged with estradiol at a later time. The effect was most pronounced for days 2 and 3. The possibility that this early effect of estradiol may involve other mechanisms than those operative in the estradiol action at later stages, is discussed.

RESUMEN

Using neonatal and immature female NMRI mice, age related variations in histochemically demonstrable adenylate cyclase activity were studied in the vaginal epithelium. Basal enzyme activity increased from day 3 to 6 after birth, only later to decrease. Almost no activity was seen on day 14. An estradiol induced increase in enzyme activity occurred in 3- and 6-day-old animals but not in 14-day-old animals. In spite of this difference both neonatal and 14-day-old animals respond to estradiol with an increased amount of a cellular product with antigenic properties specific for the cervicovaginal epithelium. The results are discussed in relation to estrogen induced irreversible effects in the neonatal cervicovaginal epithelium.