Interplay between heat shock proteins HSP101 and HSA32 prolongs heat acclimation memory posttranscriptionally in Arabidopsis.

Heat acclimation improves the tolerance of organisms to severe heat stress. Our previous work showed that in Arabidopsis (Arabidopsis thaliana), the "memory" of heat acclimation treatment decayed faster in the absence of the heat-stress-associated 32-kD protein HSA32, a heat-induced protein predominantly found in plants. The HSA32 null mutant attains normal short-term acquired thermotolerance but is defective in long-term acquired thermotolerance. To further explore this phenomenon, we isolated Arabidopsis defective in long-term acquired thermotolerance (dlt) mutants using a forward genetic screen. Two recessive missense alleles, dlt1-1 and dlt1-2, encode the molecular chaperone heat shock protein101 (HSP101). Results of immunoblot analyses suggest that HSP101 enhances the translation of HSA32 during recovery after heat treatment, and in turn, HSA32 retards the decay of HSP101. The dlt1-1 mutation has little effect on HSP101 chaperone activity and thermotolerance function but compromises the regulation of HSA32. In contrast, dlt1-2 impairs the chaperone activity and thermotolerance function of HSP101 but not the regulation of HSA32. These results suggest that HSP101 has a dual function, which could be decoupled by the mutations. Pulse-chase analysis showed that HSP101 degraded faster in the absence of HSA32. The autophagic proteolysis inhibitor E-64d, but not the proteasome inhibitor MG132, inhibited the degradation of HSP101. Ectopic expression of HSA32 confirmed its effect on the decay of HSP101 at the posttranscriptional level and showed that HSA32 was not sufficient to confer long-term acquired thermotolerance when the HSP101 level was low. Taken together, we propose that a positive feedback loop between HSP101 and HSA32 at the protein level is a novel mechanism for prolonging the memory of heat acclimation.

A consensus envelope protein domain III can induce neutralizing antibody responses against serotype 2 of dengue virus in non-human primates.

We have previously demonstrated that vaccination with a subunit dengue vaccine containing a consensus envelope domain III with aluminum phosphate elicits neutralizing antibodies against all four serotypes of dengue virus in mice. In this study, we evaluated the immunogenicity of the subunit dengue vaccine in non-human primates. After vaccination, monkeys that received the subunit vaccine with aluminum phosphate developed a significantly strong and long-lasting antibody response. A specific T cell response with cytokine production was also induced, and this correlated with the antibody response. Additionally, neutralizing antibodies against serotype 2 were detected in two of three monkeys. The increase in serotype-2-specific antibody titers and avidity observed in these two monkeys suggested that a serotype-2-biased antibody response occurs. These data provide evidence that a protective neutralizing antibody response was successfully elicited in non-human primates by the dengue subunit vaccine with aluminum phosphate adjuvant.

Microbiota signatures associated with invasive Candida albicans infection in the gastrointestinal tract of immunodeficient mice.

Candida albicans is a commensal microorganism in the human gut but occasionally causes invasive C. albicans infection (ICA), especially in immunocompromised individuals. Early initiation of antifungal therapy is associated with reduced mortality of ICA, but rapid diagnosis remains a challenge. The ICA-associated changes in the gut microbiota can be used as diagnostic and therapeutic targets but have been poorly investigated. In this study, we utilized an immunodeficient Rag2γc (Rag2-/-il2γc-/-) mouse model to investigate the gut microbiota alterations caused by C. albicans throughout its cycle, from its introduction into the gastrointestinal tract to invasion, in the absence of antibiotics. We observed a significant increase in the abundance of Firmicutes, particularly Lachnospiraceae and Ruminococcaceae, as well as a significant decrease in the abundance of Candidatus Arthromitus in mice exposed to either the wild-type SC5314 strain or the filamentation-defective mutant (cph1/cph1 efg1/efg1) HLC54 strain of C. albicans. However, only the SC5314-infected mice developed ICA. A linear discriminate analysis of the temporal changes in the gut bacterial composition revealed Bacteroides vulgatus as a discriminative biomarker associated with SC5314-infected mice with ICA. Additionally, a positive correlation between the B. vulgatus abundance and fungal load was found, and the negative correlation between the Candidatus Arthromitus abundance and fungal load after exposure to C. albicans suggested that C. albicans might affect the differentiation of intestinal Th17 cells. Our findings reveal the influence of pathogenic C. albicans on the gut microbiota and identify the abundance of B. vulgatus as a microbiota signature associated with ICA in an immunodeficient mouse model.

Candida albicans Colonizes and Disseminates to the Gastrointestinal Tract in the Presence of the Microbiota in a Severe Combined Immunodeficient Mouse Model.

Candida albicans is the leading cause of candidemia or other invasive candidiasis. Gastrointestinal colonization has been considered as the primary source of candidemia. However, few established mouse models that mimic this infection route are available. In the present study, we established a mouse model of disseminated candidiasis developed through the translocation of Candida from the gut. In this study, we developed a novel C. albicans GI colonization and dissemination animal model by using severe combined immunodeficient Rag2-/-IL2γc-/- (Rag2γc) mice, which lack functional T, B, NK cells, and IL2γc-dependent signaling. Rag2γc mice were highly susceptible to C. albicans gastrointestinal infection even in the presence of the gut microbiota. Within 4 weeks post infection, Rag2γc mice showed dose-dependent weight loss and disseminated candidiasis in more than 58% (7/12) of moribund mice. Histological analysis demonstrated abundant hyphae penetrating the mucosa, with significant neutrophilic infiltration in mice infected with wild-type C. albicans but not a filamentation-defective mutant. In moribund Rag2γc mice, the necrotic lesions and disrupted epithelial cells were associated with C. albicans hyphae. Notably, removal of the gut microbiota by antibiotics exacerbated the severity of fungal infection in Rag2γc mice, as demonstrated by elevated fungal burdens and accelerated weight loss and death. Furthermore, higher fungal burden and IL-1ß expression were prominently noted in the stomach of Rag2γc mice. In fact, a significant increase in circulating proinflammatory cytokines, including IL-6, TNF-α, and IL-10, indicative of a septic response, was evident in infected Rag2γc mice. Additionally, Rag2γc mice exhibited significantly lower levels of IL-22 but not IFN-γ or IL-17A than wild-type B6 mice, suggesting that IL-22 plays a role in C. albicans gastrointestinal infection. Collectively, our analysis of the Rag2γc mouse model revealed features of C. albicans gastrointestinal colonization and dissemination without the interference from antibiotics or chemotherapeutic agents, thus offering a new investigative tool for delineating the pathogenesis of C. albicans and its cross-talk with the gut microbiota.

Long-term immunogenicity studies of formalin-inactivated enterovirus 71 whole-virion vaccine in macaques.

Enterovirus 71 (EV71) has caused epidemics of hand, foot and mouth diseases in Asia during the past decades and no vaccine is available. A formalin-inactivated EV71 candidate vaccine (EV71vac) based on B4 subgenotype has previously been developed and found to elicit strong neutralizing antibody responses in mice and humans. In this study, we evaluated the long-term immunogenicity and safety of this EV71vac in a non-human primate model. Juvenile macaques were immunized at 0, 3 and 6 weeks either with 10 or 5 µg doses of EV71vac formulated with AlPO4 adjuvant, or PBS as control. During the 56 weeks of studies, no fever nor local redness and swelling at sites of injections was observed in the immunized macaques. After single immunization, 100% seroconversion based on 4-fold increased in neutralization titer (Nt) was detected in EV71vac immunized monkeys but not PBS controls. A dose-dependent IgG antibody response was observed in monkeys receiving EV71vac immunization. The Nt of EV71vac immunized macaques had reached the peak after 3 vaccinations, then decreased gradually; however, the GMT of neutralizing antibody in the EV71vac immunized macaques were still above 100 at the end of the study. Correspondingly, both dose- and time-dependent interferon-γ and CD4+ T cell responses were detected in monkeys receiving EV71vac. Interestingly, similar to human responses, the dominant T cell epitopes of macaques were identified mainly in VP2 and VP3 regions. In addition, strong cross-neutralizing antibodies against most EV71 subgenotypes except some C2 and C4b strains, and Coxsackievirus A16 were observed. In summary, our results indicate that EV71vac elicits dose-dependent T-cell and antibody responses in macaques that could be a good animal model for evaluating the long-term immune responses elicited by EV71 vaccines.