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.

Induction of high affinity monoclonal antibodies against SARS-CoV-2 variant infection using a DNA prime-protein boost strategy.

BACKGROUND: Calls for the coronavirus to be treated as an endemic illness, such as the flu, are increasing. After achieving high coverage of COVID-19 vaccination, therapeutic drugs have become important for future SARS-CoV-2 variant outbreaks. Although many monoclonal antibodies have been approved for emergency use as treatments for SARS-CoV-2 infection, some monoclonal antibodies are not authorized for variant treatment. Broad-spectrum monoclonal antibodies are unmet medical needs. METHODS: We used a DNA prime-protein boost approach to generate high-quality monoclonal antibodies. A standard ELISA was employed for the primary screen, and spike protein-human angiotensin-converting enzyme 2 blocking assays were used for the secondary screen. The top 5 blocking clones were selected for further characterization, including binding ability, neutralization potency, and epitope mapping. The therapeutic effects of the best monoclonal antibody against SARS-CoV-2 infection were evaluated in a hamster infection model. RESULTS: Several monoclonal antibodies were selected that neutralize different SARS-CoV-2 variants of concern (VOCs). These VOCs include Alpha, Beta, Gamma, Delta, Kappa and Lambda variants. The high neutralizing antibody titers against the Beta variant would be important to treat Beta-like variants. Among these monoclonal antibodies, mAb-S5 displays the best potency in terms of binding affinity and neutralizing capacity. Importantly, mAb-S5 protects animals from SARS-CoV-2 challenge, including the Wuhan strain, D614G, Alpha and Delta variants, although mAb-S5 exhibits decreased neutralization potency against the Delta variant. Furthermore, the identified neutralizing epitopes of monoclonal antibodies are all located in the receptor-binding domain (RBD) of the spike protein but in different regions. CONCLUSIONS: Our approach generates high-potency monoclonal antibodies against a broad spectrum of VOCs. Multiple monoclonal antibody combinations may be the best strategy to treat future SARS-CoV-2 variant outbreaks.

Immunodomination of Serotype-Specific CD4+ T-Cell Epitopes Contributed to the Biased Immune Responses Induced by a Tetravalent Measles-Vectored Dengue Vaccine.

Dengue is an emerging mosquito-borne disease, and the use of prophylactic vaccines is still limited. We previously developed a tetravalent dengue vaccine (rMV-TDV) by a recombinant measles virus (MV) vector expressing envelope protein domain III (ED3). In this study, we used dengue-susceptible AG129 mice to evaluate the protective and/or pathogenic immune responses induced by rMV-TDV. Consistent with the previous study, rMV-TDV-immunized mice developed a significant neutralizing antibody response against all serotypes of DENV, as well as a significant IFN-γ response biased to DENV-3, compared to the vector controls. We further demonstrated that this DENV-3-specific IFN-γ response was dominated by one CD4+ T-cell epitope located in E349-363. After DENV-2 challenge, rMV-TDV-immunized mice showed a significantly lower viremia and no inflammatory cytokine increase compared to the vector controls, which had an ~100 times higher viremia and a significant increase in IFN-γ and TNF-α. As a correlate of protection, a robust memory IFN-γ response specific to DENV-2 was boosted in rMV-TDV-immunized mice after challenge. This result suggested that pre-existing DENV-3-dominated T-cell responses did not cross-react, but a DENV-2-specific IFN-γ response, which was undetectable during immunization, was recalled. Interestingly, this recalled T-cell response recognized the epitope in the same position as the E349-363 but in the DENV-2 serotype. This result suggested that immunodomination occurred in the CD4+ T-cell epitopes between dengue serotypes after rMV-TDV vaccination and resulted in a DENV-3-dominated CD4+ T-cell response. Although the significant increase in IgG against both DENV-2 and -3 suggested that cross-reactive antibody responses were boosted, the increased neutralizing antibodies and IgG avidity still remained DENV-2 specific, consistent with the serotype-specific T cell response post challenge. Our data reveal that immunodomination caused a biased T-cell response to one of the dengue serotypes after tetravalent dengue vaccination and highlight the roles of cross-reactive T cells in dengue protection.

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.

DUSP6 mediates T cell receptor-engaged glycolysis and restrains TFH cell differentiation.

Activated T cells undergo metabolic reprogramming and effector-cell differentiation but the factors involved are unclear. Utilizing mice lacking DUSP6 (DUSP6-/-), we show that this phosphatase regulates T cell receptor (TCR) signaling to influence follicular helper T (TFH) cell differentiation and T cell metabolism. In vitro, DUSP6-/- CD4+ TFH cells produced elevated IL-21. In vivo, TFH cells were increased in DUSP6-/- mice and in transgenic OTII-DUSP6-/- mice at steady state. After immunization, DUSP6-/- and OTII-DUSP6-/- mice generated more TFH cells and produced more antigen-specific IgG2 than controls. Activated DUSP6-/- T cells showed enhanced JNK and p38 phosphorylation but impaired glycolysis. JNK or p38 inhibitors significantly reduced IL-21 production but did not restore glycolysis. TCR-stimulated DUSP6-/- T cells could not induce phosphofructokinase activity and relied on glucose-independent fueling of mitochondrial respiration. Upon CD28 costimulation, activated DUSP6-/- T cells did not undergo the metabolic commitment to glycolysis pathway to maintain viability. Unexpectedly, inhibition of fatty acid oxidation drastically lowered IL-21 production in DUSP6-/- TFH cells. Our findings suggest that DUSP6 connects TCR signaling to activation-induced metabolic commitment toward glycolysis and restrains TFH cell differentiation via inhibiting IL-21 production.

Detection and quantification of dengue virus using a novel biosensor system based on dengue NS3 protease activity.

BACKGROUND: The traditional methods, plaque assays and immuno-focus assays, used to titrate infectious dengue virus (DENV) particles are time consuming and labor intensive. Here, we developed a DENV protease activity detection system (DENPADS) to visualize DENV infection in cells based on dengue protease activity. METHODOLOGY/PRINCIPAL FINDINGS: Dengue NS3 protease cleaves NS4B-NS5. BHK-21 cells stably expressing the sensor module comprising DENV-2 NS4 and the 10 amino-terminal amino acids of NS5 (N10NS5) fused with the SV40 nuclear localization signal (NLS) and Cre recombinase (Cre), were generated. Cre is constrained outside the nucleus in the absence of NS3 activity but translocates into the nucleus through NS4B-NS5 cleavage when cells are infected with DENV. Nuclear translocation of Cre can trigger the reporter system, which contains a cis-loxP-flanked mCherry with three continuous stop codons following an SV40 polyA tail cDNA upstream of EGFP or mHRP cDNA. Our results show that DENPADS is an efficient and accurate method to titrate 4 DENV serotypes in 24 hours. Compared with current virus titration methods, the entire process is easy to perform, and the data are easily acquired. CONCLUSIONS/SIGNIFICANCE: In this study, we demonstrate that DENPADS can be used to detect dengue viral infection through a fluorescence switch or HRP activity in the infected cells. This approach is sensitive with less incubation time and labor input. In addition, DENPADS can simultaneously evaluate the efficacy and cytotoxicity of potential anti-DENV candidates. Overall, DENPADS is a useful tool for dengue research.

Immunogenicity of a novel tetravalent vaccine formulation with four recombinant lipidated dengue envelope protein domain IIIs in mice.

We developed a novel platform to express high levels of recombinant lipoproteins with intrinsic adjuvant properties. Based on this technology, our group developed recombinant lipidated dengue envelope protein domain IIIs as vaccine candidates against dengue virus. This work aims to evaluate the immune responses in mice to the tetravalent formulation. We demonstrate that 4 serotypes of recombinant lipidated dengue envelope protein domain III induced both humoral and cellular immunity against all 4 serotypes of dengue virus on the mixture that formed the tetravalent formulation. Importantly, the immune responses induced by the tetravalent formulation in the absence of the exogenous adjuvant were functional in clearing the 4 serotypes of dengue virus in vivo. We affirm that the tetravalent formulation of recombinant lipidated dengue envelope protein domain III is a potential vaccine candidate against dengue virus and suggest further detailed studies of this formulation in nonhuman primates.

The successful induction of T-cell and antibody responses by a recombinant measles virus-vectored tetravalent dengue vaccine provides partial protection against dengue-2 infection.

Dengue has a major impact on global public health, and the use of dengue vaccine is very limited. In this study, we evaluated the immunogenicity and protective efficacy of a dengue vaccine made from a recombinant measles virus (MV) that expresses envelope protein domain III (ED3) of dengue-1 to 4. Following immunization with the MV-vectored dengue vaccine, mice developed specific interferon-gamma and antibody responses against dengue virus and MV. Neutralizing antibodies against MV and dengue viruses were also induced, and protective levels of FRNT50 ≥ 10 to 4 serotypes of dengue viruses were detected in the MV-vectored dengue vaccine-immunized mice. In addition, specific interferon-gamma and antibody responses to dengue viruses were still induced by the MV-vectored dengue vaccine in mice that were pre-infected with MV. This finding suggests that the pre-existing immunity to MV did not block the initiation of immune responses. By contrast, mice that were pre-infected with dengue-3 exhibited no effect in terms of their antibody responses to MV and dengue viruses, but a dominant dengue-3-specific T-cell response was observed. After injection with dengue-2, a detectable but significantly lower viremia and a higher titer of anti-dengue-2 neutralizing antibodies were observed in MV-vectored dengue vaccine-immunized mice versus the vector control, suggesting that an anamnestic antibody response that provided partial protection against dengue-2 was elicited. Our results with regard to T-cell responses and the effect of pre-immunity to MV or dengue viruses provide clues for the future applications of an MV-vectored dengue vaccine.

The Immunodominance Change and Protection of CD4+ T-Cell Responses Elicited by an Envelope Protein Domain III-Based Tetravalent Dengue Vaccine in Mice.

Dengue is the leading cause of mosquito-borne viral infections and no vaccine is available now. Envelope protein domain III (ED3) is the major target for the binding of dengue virus neutralizing antibodies; however, the ED3-specifc T-cell response is less well understood. To investigate the T-cell responses to four serotypes of dengue virus (DENV-1 to 4), we immunized mice using either a tetravalent ED3-based DNA or protein vaccine, or combined both as a DNA prime-protein boost strategy (prime-boost). A significant serotype-dependent IFN-γ or IL-4 response was observed in mice immunized with either the DNA or protein vaccine. The IFN-γ response was dominant to DENV-1 to 3, whereas the IL-4 response was dominant to DENV-4. Although the similar IgG titers for the four serotypes were observed in mice immunized with the tetravalent vaccines, the neutralizing antibody titers varied and followed the order of 2 = 3>1>4. Interestingly, the lower IFN-γ response to DENV-4 is attributable to the immunodominance change between two CD4+ T-cell epitopes; one T-cell epitope located at E349-363 of DENV-1 to 3 was more immunogenic than the DENV-4 epitope E313-327. Despite DENV-4 specific IFN-γ responses were suppressed by immunodominance change, either DENV-4-specific IFN-γ or neutralizing antibody responses were still recalled after DENV-4 challenge and contributed to virus clearance. Immunization with the prime-boost elicited both IFN-γ and neutralizing antibody responses and provided better protection than either DNA or protein immunization. Our findings shed light on how ED3-based tetravalent dengue vaccines sharpen host CD4 T-cell responses and contribute to protection against dengue virus.

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.

A poorly neutralizing IgG2a/c response elicited by a DNA vaccine protects mice against Japanese encephalitis virus.

We demonstrated previously that immunization with a DNA vaccine expressing the Japanese encephalitis virus (JEV) envelope (E) protein conferred a high level of protection through a poorly neutralizing antibody response. Here, we further investigated the role of the IgG subclass in this antibody-dependent protection using cytokine co-immunization and cytokine-deficient mice. A significant difference in IgG2a/c but not IgG1 was observed between mice that survived or died following a lethal challenge. Correspondingly, the IgG2a/c response and protection increased in IL-4-deficient mice but decreased in IFN-γ-deficient mice, highlighting the importance of IgG2a/c. In addition, the restoration of protection and E-specific IgG2a/c production in IFN-γ-deficient mice by a T helper (Th) type 1-biased intramuscular immunization suggested that IgG2a/c but not IFN-γ was the major component for protection. The failure of protection against a direct intracranial challenge indicated that IgG2a/c-mediated protection was restricted to outside the central nervous system. Consistent with this conclusion, passive transfer of E-specific antisera conferred protection only pre-exposure to JEV. Therefore, our data provided evidence that the IgG subclass plays an important role in protection against JEV, particular in poorly neutralizing E-specific antibodies, and Th1-biased IgG2a/c confers better protection than Th2-biased IgG1 against JEV.

Vaccine-induced measles virus-specific T cells do not prevent infection or disease but facilitate subsequent clearance of viral RNA.

Infection with wild-type measles virus (MeV) induces lifelong protection from reinfection, and parenteral delivery of the live attenuated measles vaccine (LAV) also provides protection from measles. The level of neutralizing antibody is a good indicator of protection, but the independent roles of MeV-specific antibody and T cells have not been identified. In this study, macaques immunized with LAV through a nebulizer and a mouthpiece developed MeV-specific T-cell responses but not neutralizing antibodies. Upon challenge with wild-type MeV, these animals developed rashes and viremias similar to those in naive animals but cleared viral RNA from blood 25 to 40 days faster. The nebulizer-immunized animals also had more robust MeV-specific CD4(+) and CD8(+) T-cell responses than the naive animals after challenge, characterized by a higher number and better durability of gamma interferon (IFN-γ)-producing cells. Induction of MeV-specific circulating CD4(+) and CD8(+) T cells capable of producing multiple cytokines correlated with clearance of viral RNA in the nebulizer-immunized macaques. These studies demonstrated that MeV-specific T-cell immunity alone did not prevent measles, but T-cell priming enhanced the magnitude, durability, and polyfunctionality of MeV-specific T cells after challenge infection and correlated with more rapid clearance of MeV RNA. IMPORTANCE The components of vaccine-induced immunity necessary for protection from infection and disease have not been clearly identified for most vaccines. Vaccine development usually focuses on induction of antibody, but T-cell-based vaccines are also under development. The live attenuated measles vaccine (LAV) given subcutaneously induces both T cells and neutralizing antibody and provides solid protection from infection. LAV delivered to the upper respiratory tract through a nebulizer and mouthpiece induced a T-cell response but no neutralizing antibody. These T-cell-primed macaques demonstrated no protection from rash or viremia when challenged with wild-type MeV, but viral RNA was cleared more rapidly than in unimmunized animals. Thus, T-cell immunity did not protect from infection or acute disease but facilitated virus clearance during recovery. These studies demonstrate the importance and independent roles of T cells and antibody in protection and recovery from measles.

Lipidated dengue-2 envelope protein domain III independently stimulates long-lasting neutralizing antibodies and reduces the risk of antibody-dependent enhancement.

BACKGROUND: Dengue virus is a mosquito-transmitted virus that can cause self-limiting dengue fever, severe life-threatening dengue hemorrhagic fever and dengue shock syndrome. The existence of four serotypes of dengue virus has complicated the development of an effective and safe dengue vaccine. Recently, a clinical phase 2b trial of Sanofi Pasteur's CYD tetravalent dengue vaccine revealed that the vaccine did not confer full protection against dengue-2 virus. New approaches to dengue vaccine development are urgently needed. Our approach represents a promising method of dengue vaccine development and may even complement the deficiencies of the CYD tetravalent dengue vaccine. METHODOLOGY/PRINCIPAL FINDINGS: Two important components of a vaccine, the immunogen and immunopotentiator, were combined into a single construct to generate a new generation of vaccines. We selected dengue-2 envelope protein domain III (D2ED III) as the immunogen and expressed this protein in lipidated form in Escherichia coli, yielding an immunogen with intrinsic immunopotentiation activity. The formulation containing lipidated D2ED III (LD2ED III) in the absence of exogenous adjuvant elicited higher D2ED III-specific antibody responses than those obtained from its nonlipidated counterpart, D2ED III, and dengue-2 virus. In addition, the avidity and neutralizing capacity of the antibodies induced by LD2ED III were higher than those elicited by D2ED III and dengue-2 virus. Importantly, we showed that after lipidation, the subunit candidate LD2ED III exhibited increased immunogenicity while reducing the potential risk of antibody-dependent enhancement of infection in mice. CONCLUSIONS/SIGNIFICANCE: Our study suggests that the lipidated subunit vaccine approach could be applied to other serotypes of dengue virus and other pathogens.

Induction of robust immunity by the emulsification of recombinant lipidated dengue-1 envelope protein domain III.

Many attempts have focused on the use of either immunomodulators or antigen delivery systems to obtain an efficacious vaccine. Here, we report a novel approach that combined an immunomodulator and delivery system to enhance antigen association and induce robust immunity. We expressed a recombinant lipidated dengue-1 envelope protein domain III (LD1ED III) and its non-lipidated form, D1ED III, in an Escherichia coli system. The LD1ED III contains a bacterial lipid moiety, which is a potent immunomodulator. We demonstrated that LD1ED III possesses an inherent immunostimulation ability that can activate RAW 264.7 macrophage cells by up-regulating their expression of CD40, CD80, CD83, CD86 and MHC II, whereas D1ED III could not induce the up-regulation of these molecules. Moreover, combining LD1ED III with a multiphase emulsion system (called PELC) increased the antigen association more than either combining D1ED III with PELC or the antigen alone. Enhanced antigen association has been shown to correlate with stronger T cell responses, greater antibody avidity and improved neutralizing capacity. Our results demonstrate that combining recombinant lipoproteins with PELC improved both the intensity and the quality of the immune response. This approach is a promising strategy for the development of subunit vaccines that induce robust immunity.

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.

Measles virus, immune control, and persistence.

Measles remains one of the most important causes of child morbidity and mortality worldwide with the greatest burden in the youngest children. Most acute measles deaths are owing to secondary infections that result from a poorly understood measles-induced suppression of immune responses. Young children are also vulnerable to late development of subacute sclerosing panencephalitis, a progressive, uniformly fatal neurologic disease caused by persistent measles virus (MeV) infection. During acute infection, the rash marks the appearance of the adaptive immune response and CD8(+) T cell-mediated clearance of infectious virus. However, after clearance of infectious virus, MeV RNA persists and can be detected in blood, respiratory secretions, urine, and lymphoid tissue for many weeks to months. This prolonged period of virus clearance may help to explain measles immunosuppression and the development of lifelong immunity to re-infection, as well as occasional infection of the nervous system. Once MeV infects neurons, the virus can spread trans-synaptically and the envelope proteins needed to form infectious virus are unnecessary, accumulate mutations, and can establish persistent infection. Identification of the immune mechanisms required for the clearance of MeV RNA from multiple sites will enlighten our understanding of the development of disease owing to persistent infection.

Comparison of the immune responses induced by chimeric alphavirus-vectored and formalin-inactivated alum-precipitated measles vaccines in mice.

A variety of vaccine platforms are under study for development of new vaccines for measles. Problems with past measles vaccines are incompletely understood and underscore the need to understand the types of immune responses induced by different types of vaccines. Detailed immune response evaluation is most easily performed in mice. Although mice are not susceptible to infection with wild type or vaccine strains of measles virus, they can be used for comparative evaluation of the immune responses to measles vaccines of other types. In this study we compared the immune responses in mice to a new protective alphavirus replicon particle vaccine expressing the measles virus hemagglutinin (VEE/SIN-H) with a non-protective formalin-inactivated, alum-precipitated measles vaccine (FI-MV). MV-specific IgG levels were similar, but VEE/SIN-H antibody was high avidity IgG2a with neutralizing activity while FI-MV antibody was low-avidity IgG1 without neutralizing activity. FI-MV antibody was primarily against the nucleoprotein with no priming to H. Germinal centers appeared, peaked and resolved later for FI-MV. Lymph node MV antibody-secreting cells were more numerous after FI-MV than VEE/SIN-H, but were similar in the bone marrow. VEE/SIN-H-induced T cells produced IFN-gamma and IL-4 both spontaneously ex vivo and after stimulation, while FI-MV-induced T cells produced IL-4 only after stimulation. In summary, VEE/SIN-H induced a balanced T cell response and high avidity neutralizing IgG2a while FI-MV induced a type 2 T cell response, abundant plasmablasts, late germinal centers and low avidity non-neutralizing IgG1 against the nucleoprotein.

A chimeric alphavirus replicon particle vaccine expressing the hemagglutinin and fusion proteins protects juvenile and infant rhesus macaques from measles.

Measles remains a major cause of child mortality, in part due to an inability to vaccinate young infants with the current live attenuated virus vaccine (LAV). To explore new approaches to infant vaccination, chimeric Venezuelan equine encephalitis/Sindbis virus (VEE/SIN) replicon particles were used to express the hemagglutinin (H) and fusion (F) proteins of measles virus (MV). Juvenile rhesus macaques vaccinated intradermally with a single dose of VEE/SIN expressing H or H and F proteins (VEE/SIN-H or VEE/SIN-H+F, respectively) developed high titers of MV-specific neutralizing antibody and gamma-interferon (IFN-gamma)-producing T cells. Infant macaques vaccinated with two doses of VEE/SIN-H+F also developed neutralizing antibody and IFN-gamma-producing T cells. Control animals were vaccinated with LAV or with a formalin-inactivated measles vaccine (FIMV). Neutralizing antibody remained above the protective level for more than 1 year after vaccination with VEE/SIN-H, VEE/SIN-H+F, or LAV. When challenged with wild-type MV 12 to 17 months after vaccination, all vaccinated juvenile and infant monkeys vaccinated with VEE/SIN-H, VEE/SIN-H+F, and LAV were protected from rash and viremia, while FIMV-vaccinated monkeys were not. Antibody was boosted by challenge in all groups. T-cell responses to challenge were biphasic, with peaks at 7 to 25 days and at 90 to 110 days in all groups, except for the LAV group. Recrudescent T-cell activity coincided with the presence of MV RNA in peripheral blood mononuclear cells. We conclude that VEE/SIN expressing H or H and F induces durable immune responses that protect from measles and offers a promising new approach for measles vaccination. The viral and immunological factors associated with long-term control of MV replication require further investigation.

Use of Vaxfectin adjuvant with DNA vaccine encoding the measles virus hemagglutinin and fusion proteins protects juvenile and infant rhesus macaques against measles virus.

A measles virus vaccine for infants under 6 months of age would help control measles. DNA vaccines hold promise, but none has provided full protection from challenge. Codon-optimized plasmid DNAs encoding the measles virus hemagglutinin and fusion glycoproteins were formulated with the cationic lipid-based adjuvant Vaxfectin. In mice, antibody and gamma interferon (IFN-gamma) production were increased by two- to threefold. In macaques, juveniles vaccinated at 0 and 28 days with 500 microg of DNA intradermally or with 1 mg intramuscularly developed sustained neutralizing antibody and H- and F-specific IFN-gamma responses. Infant monkeys developed sustained neutralizing antibody and T cells secreting IFN-gamma and interleukin-4. Twelve to 15 months after vaccination, vaccinated monkeys were protected from an intratracheal challenge: viremia was undetectable by cocultivation and rashes did not appear, while two naïve monkeys developed viremia and rashes. The use of Vaxfectin-formulated DNA is a promising approach to the development of a measles vaccine for young infants.

Dose-dependent protection against or exacerbation of disease by a polylactide glycolide microparticle-adsorbed, alphavirus-based measles virus DNA vaccine in rhesus macaques.

Measles remains an important cause of vaccine-preventable child mortality. Development of a low-cost, heat-stable vaccine for infants under the age of 6 months could improve measles control by facilitating delivery at the time of other vaccines and by closing a window of susceptibility prior to immunization at 9 months of age. DNA vaccines hold promise for development, but achieving protective levels of antibody has been difficult and there is an incomplete understanding of protective immunity. In the current study, we evaluated the use of a layered alphavirus DNA/RNA vector encoding measles virus H (SINCP-H) adsorbed onto polylactide glycolide (PLG) microparticles. In mice, antibody and T-cell responses to PLG-formulated DNA were substantially improved compared to those to naked DNA. Rhesus macaques received two doses of PLG/SINCP-H delivered either intramuscularly (0.5 mg) or intradermally (0.5 or 0.1 mg). Antibody and T-cell responses were induced but not sustained. On challenge, the intramuscularly vaccinated monkeys did not develop rashes and had lower viremias than vector-treated control monkeys. Monkeys vaccinated with the same dose intradermally developed rashes and viremia. Monkeys vaccinated intradermally with the low dose developed more severe rashes, with histopathologic evidence of syncytia and intense dermal and epidermal inflammation, eosinophilia, and higher viremia compared to vector-treated control monkeys. Protection after challenge correlated with gamma interferon-producing T cells and with early production of high-avidity antibody that bound wild-type H protein. We conclude that PLG/SINCP-H is most efficacious when delivered intramuscularly but does not provide an advantage over standard DNA vaccines for protection against measles.