Identification of Signaling Pathways by Which CD40 Stimulates Autophagy and Antimicrobial Activity against Toxoplasma gondii in Macrophages

Identification of Signaling Pathways by Which CD40 Stimulates Autophagy and Antimicrobial Activity against Toxoplasma gondii in Macrophages

ABSTRACT

CD40 is an important stimulator of autophagy and autophagic killing of Toxoplasma gondii in host cells. In contrast to autophagy induced by nutrient deprivation or pattern recognition receptors, less is known about the effects of cell-mediated immunity on Beclin 1 and ULK1, key regulators of autophagy. Here we studied the molecular mechanisms by which CD40 stimulates autophagy in macrophages. CD40 ligation caused biphasic Jun N-terminal protein kinase (JNK) phosphorylation. The second phase of JNK phosphorylation was dependent on autocrine production of tumor necrosis factor alpha (TNF-α). TNF-α and JNK signaling were required for the CD40-induced increase in autophagy. JNK signaling downstream of CD40 caused Ser-87 phosphorylation of Bcl-2 and dissociation between Bcl-2 and Beclin 1, an event known to stimulate the autophagic function of Beclin 1. However, TNF-α alone was unable to stimulate autophagy. CD40 also stimulated autophagy via a pathway that included calcium/calmodulin-dependent kinase kinase β (CaMKKβ), AMP-activated protein kinase (AMPK), and ULK1. CD40 caused AMPK phosphorylation at its activating site, Thr-172. This effect was mediated by CaMKKβ and was not impaired by neutralization of TNF-α. CD40 triggered AMPK-dependent Ser-555 phosphorylation of ULK1. CaMKKβ, AMPK, and ULK1 were required for CD40-induced increase in autophagy. CD40-mediated autophagic killing of Toxoplasma gondii is known to require TNF-α. Knockdown of JNK, CaMKKβ, AMPK, or ULK1 prevented T. gondii killing in CD40-activated macrophages. The second phase of JNK phosphorylation—Bcl-2 phosphorylation—Bcl-2–Beclin 1 dissociation and AMPK phosphorylation-ULK1 phosphorylation occurred simultaneously at ∼4 h post-CD40 stimulation. Thus, CaMKKβ and TNF-α are upstream molecules by which CD40 acts on ULK1 and Beclin 1 to stimulate autophagy and killing of T. gondii.

Jawahar Raina
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Malaria Vaccines: Recent Advances and New Horizons

Malaria Vaccines: Recent Advances and New Horizons

Abstract

The development of highly effective and durable vaccines against the human malaria parasites Plasmodium falciparum and P. vivax remains a key priority. Decades of endeavor have taught that achieving this goal will be challenging; however, recent innovation in malaria vaccine research and a diverse pipeline of novel vaccine candidates for clinical assessment provides optimism. With first-generation pre-erythrocytic vaccines aiming for licensure in the coming years, it is important to reflect on how next-generation approaches can improve on their success. Here we review the latest vaccine approaches that seek to prevent malaria infection, disease, and transmission and highlight some of the major underlying immunological and molecular mechanisms of protection. The synthesis of rational antigen selection, immunogen design, and immunization strategies to induce quantitatively and qualitatively improved immune effector mechanisms offers promise for achieving sustained high-level protection.

Jawahar Raina
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A two-dose heterologous prime-boost vaccine regimen eliciting sustained immune responses to Ebola Zaire could support a preventive strategy for future outbreaks

A two-dose heterologous prime-boost vaccine regimen eliciting sustained immune responses to Ebola Zaire could support a preventive strategy for future outbreaks

ABSTRACT

The consequences of the 2013–16 Ebola Zaire virus disease epidemic in West Africa were grave. The economies, healthcare systems and communities of Guinea, Sierra Leone and Liberia were devastated by over 18 months of active Ebola virus transmission, followed by sporadic resurgences potentially related to sexual transmission by survivors with viral persistence in body fluids following recovery. The need to develop and implement strategies to prevent and mitigate future outbreaks is now beyond dispute. The potential for unpredictable outbreaks of indeterminate duration, and control challenges posed by the possibility of sporadic re-emergence, mean that implementation of an effective vaccination program for outbreak containment necessitates a vaccine providing durable immunity. Heterologous prime-boost vaccine regimens deliver the same or similar antigens through different vaccine types, the first to prime and the second to boost the immune system. Ad26.ZEBOV/MVA-BN-Filo is an investigational Ebola Zaire vaccine regimen that uses this heterologous prime-boost approach. Preliminary Phase 1 data suggest that Ad26.ZEBOV/MVA-BN-Filo confers durable immunity for at least 240 d and is well-tolerated with a good safety profile. This regimen may therefore be suitable for prophylactic use in a regional or targeted population vaccination strategy, and could potentially aid prevention and control of future Ebola outbreaks.

KEYWORDS: Ebola virus, heterologous prime-boost, Ad26.ZEBOV/MVA-BN-Filo, viral persistence, sustained immunity, population vaccination strategy
Jawahar Raina
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High Zika Virus Seroprevalence in Salvador, Northeastern Brazil Limits the Potential for Further Outbreaks

High Zika Virus Seroprevalence in Salvador, Northeastern Brazil Limits the Potential for Further Outbreaks

ABSTRACT

During 2015 to 2016, Brazil reported more Zika virus (ZIKV) cases than any other country, yet population exposure remains unknown. Serological studies of ZIKV are hampered by cross-reactive immune responses against heterologous viruses. We conducted serosurveys for ZIKV, dengue virus (DENV), and Chikungunya virus (CHIKV) in 633 individuals prospectively sampled during 2015 to 2016, including microcephaly and non-microcephaly pregnancies, HIV-infected patients, tuberculosis patients, and university staff in Salvador in northeastern Brazil using enzyme-linked immunosorbent assays (ELISAs) and plaque reduction neutralization tests. Sera sampled retrospectively during 2013 to 2015 from 277 HIV-infected patients were used to assess the spread of ZIKV over time. Individuals were georeferenced, and sociodemographic indicators were compared between ZIKV-positive and -negative areas and areas with and without microcephaly cases. Epidemiological key parameters were modeled in a Bayesian framework. ZIKV seroprevalence increased rapidly during 2015 to 2016, reaching 63.3% by 2016 (95% confidence interval [CI], 59.4 to 66.8%), comparable to the seroprevalence of DENV (75.7%; CI, 69.4 to 81.1%) and higher than that of CHIKV (7.4%; CI, 5.6 to 9.8%). Of 19 microcephaly pregnancies, 94.7% showed ZIKV IgG antibodies, compared to 69.3% of 257 non-microcephaly pregnancies (P = 0.017). Analyses of sociodemographic data revealed a higher ZIKV burden in low socioeconomic status (SES) areas. High seroprevalence, combined with case data dynamics allowed estimates of the basic reproduction number R0 of 2.1 (CI, 1.8 to 2.5) at the onset of the outbreak and an effective reproductive number Reff of <1 in subsequent years. Our data corroborate ZIKV-associated congenital disease and an association of low SES and ZIKV infection and suggest that population immunity caused cessation of the outbreak. Similar studies from other areas will be required to determine the fate of the American ZIKV outbreak.

KEYWORDS: Zika virus, microcephaly, risk factors, serology, socioeconomic status
Jawahar Raina
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Western Blot Detection of Human Anti-Chikungunya Virus Antibody with Recombinant Envelope 2 Protein

Western Blot Detection of Human Anti-Chikungunya Virus Antibody with Recombinant Envelope 2 Protein

Abstract

Chikungunya virus (CHIKV), a tropical pathogen, has re-emerged and has massive outbreaks abruptly all over the world. Containing many dominant epitopes, the envelope E2 protein of CHIKV has been explored for the vaccination or diagnosis. In the present study, the antigenicity of a recombinant expressed intrinsically disorder domain (IUD) of E2 was tested for the detection of the antibody against CHIKV through western blot method. The gene of the IUD of E2 was inserted into 2 different vectors and expressed as recombinant GST-E2 and recombinant MBP-E2 fusion protein, respectively. Two kinds of fusion proteins were tested with 30 CHIKV patient sera and 30 normal sera, respectively. Both proteins were detected by 25 patients sera (83.3%) and 1 normal serum (3.3%). This test showed a relatively high sensitivity and very high specificity of the recombinant E2 proteins to be used as diagnostic antigens against CHIKV infection.

Keywords: Chikungunya virus (CHIKV), envelope E2 protein, intrinsically unstructured domain (IUD), GST-E2, MBP-E2, western blot, patient sera
Jawahar Raina
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Synergistic malaria vaccine combinations identified by systematic antigen screening

Synergistic malaria vaccine combinations identified by systematic antigen screening

SIGNIFICANCE

Malaria still kills hundreds of thousands of children each year. Malaria vaccine development is complicated by high levels of parasite genetic diversity, which makes single target vaccines vulnerable to the development of variant-specific immunity. To overcome this hurdle, we systematically screened a panel of 29 blood-stage antigens from the most deadly human malaria parasite, Plasmodium falciparum. We identified several targets that were able to inhibit erythrocyte invasion in two genetically diverse strains. Testing these targets in combination identified several pairs that blocked invasion more effectively in combination than in isolation. Video microscopy and studies of natural immune responses to malaria in patients suggest that targeting multiple steps in invasion is more likely to produce a synergistic vaccine response.

Keywords: malaria, vaccine, Plasmodium falciparum, antigen combinations, erythrocyte invasion

ABSTRACT

A highly effective vaccine would be a valuable weapon in the drive toward malaria elimination. No such vaccine currently exists, and only a handful of the hundreds of potential candidates in the parasite genome have been evaluated. In this study, we systematically evaluated 29 antigens likely to be involved in erythrocyte invasion, an essential developmental stage during which the malaria parasite is vulnerable to antibody-mediated inhibition. Testing antigens alone and in combination identified several strain-transcending targets that had synergistic combinatorial effects in vitro, while studies in an endemic population revealed that combinations of the same antigens were associated with protection from febrile malaria. Video microscopy established that the most effective combinations targeted multiple discrete stages of invasion, suggesting a mechanistic explanation for synergy. Overall, this study both identifies specific antigen combinations for high-priority clinical testing and establishes a generalizable approach that is more likely to produce effective vaccines.

Jawahar Raina
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Geographical distribution of primary & secondary dengue cases in India – 2017: A cross-sectional multicentric study

Abstract

Background & objectives:

Dengue virus infection is endemic in India with all the four serotypes of dengue virus in circulation. This study was aimed to determine the geographic distribution of the primary and secondary dengue cases in India.

Methods:

A multicentre cross-sectional study was conducted at Department of Health Research / Indian Council of Medical Research (DHR)/(ICMR) viral research and diagnostic laboratories (VRDLs) and selected ICMR institutes located in India. Only laboratory-confirmed dengue cases with date of onset of illness less than or equal to seven days were included between September and October 2017. Dengue NS1 antigen ELISA and anti-dengue IgM capture ELISA were used to diagnose dengue cases while anti-dengue IgG capture ELISA was used for identifying the secondary dengue cases.

Results:

Of the 1372 dengue cases, 897 (65%) were classified as primary dengue and 475 (35%) as secondary dengue cases. However, the proportion varied widely geographically, with Theni, Tamil Nadu; Tirupati, Andhra Pradesh and Udupi-Manipal, Karnataka reporting more than 65 per cent secondary dengue cases while Srinagar, Jammu and Kashmir reporting as low as 10 per cent of the same. The median age of primary dengue cases was 25 yr [interquartile range (IQR 17-35] while that of secondary dengue cases was 23 yr (IQR 13.5-34). Secondary dengue was around 50 per cent among the children belonging to the age group 6-10 yr while it ranged between 20-43 per cent among other age groups.

Interpretation & conclusions:

Our findings showed a wide geographical variation in the distribution of primary and secondary dengue cases in India. It would prove beneficial to include primary and secondary dengue differentiation protocol in the national dengue surveillance programme.

Keywords: Dengue, geographic variation, India, primary, secondary, viral research and diagnostic laboratories
Jawahar Raina
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Secretory Microneme Proteins Induce T-Cell Recall Responses in Mice Chronically Infected with Toxoplasma gondii

Secretory Microneme Proteins Induce T-Cell Recall Responses in Mice Chronically Infected with Toxoplasma gondii

ABSTRACT

Microneme (MIC) proteins play important roles in the recognition, adhesion, and invasion of host cells by Toxoplasma gondii. Previous studies have shown that MIC proteins are highly immunogenic in the mouse and recognized by human serum antibodies. Here we report that T. gondii antigens MIC1, MIC3, MIC4, and MIC6 were capable of inducing memory responses leading to production of gamma interferon (IFN-γ) by T cells from T. gondii-infected mice. Production of IFN-γ was demonstrated using enzyme-linked immunosorbent spot (ELISPOT) assay and also intracellular cytokine staining. All four MIC antigens displayed very high sensitivity (100%) and specificity (86 to 100%) for detecting chronic infection. Interestingly, IFN-γ was produced by both CD4+ and CD8+ T cells in BALB/c mice but primarily by CD4+ T cells in C57BL/6 mice. Phenotypic characterization of IFN-γ-producing CD4+ and CD8+ T cells in BALB/c mice and CD4+ T cells in C57BL/6 mice revealed effector memory T cells (CD44hi CD62Llo) as the predominant cells that contributed to IFN-γ production in response to MIC antigens. Effector memory responses were seen in mice of different major histocompatibility complex class II (MHC-II) haplotypes, suggesting that MIC antigens contain epitopes that are broadly recognized.

IMPORTANCE Current diagnosis of toxoplasmosis relies almost exclusively on antibody detection, and while detection of IgG provides a useful estimate of prior infection, it does not alone indicate immune status. In contrast, detection of IFN-γ responses to T. gondii antigens has been used to monitor immune responsiveness in HIV-infected patients, thus providing valuable predictions about the potential for disease reactivation. However, specific T. gondii antigens that can be used in assays to detect cellular immunity remain largely undefined. In this study, we examined the diagnostic potential of microneme antigens of T. gondii using IFN-γ detection assays. Our findings demonstrate that MIC antigens (MIC1, MIC3, MIC4, and MIC6) elicit IFN-γ responses from memory T cells in chronically infected mice. Monitoring IFN-γ production by T cells stimulated with MIC antigens provided high sensitivity and specificity for detection of T. gondii infection in mice. Taken together, these studies suggest that microneme antigens might be useful as an adjunct to serological testing to monitor immune status during infection.

KEYWORDS: antigen-specific T cells, gamma interferon detection assays, memory T cells, microneme antigens, recall response, toxoplasmosis
Jawahar Raina
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Zika Virus Structure, Maturation, and Receptors

Zika Virus Structure, Maturation, and Receptors

Abstract

The emergence of Zika virus (ZIKV) as a major public health threat has focused research on understanding virus biology and developing a suite of strategies for disease intervention. Recent advances in cryoelectron microscopy have accelerated structure-function studies of flaviviruses and of ZIKV in particular. Structures of the mature and immature ZIKV have demonstrated its similarity with other known flaviviruses such as dengue and West Nile viruses. However, ZIKV’s unique pathobiology demands an explanation of how its structure, although similar to its flavivirus relatives, is sufficiently unique to address questions of receptor specificity, transmission, and antigenicity. Progress in defining the immunodominant epitopes and how neutralizing antibodies bind to them will provide great insight as vaccines progress through clinical trials. Identification of host receptors will substantially illuminate the interesting ZIKV tropism and provide insights into pathogenesis. Although the answers to all of these questions are not yet available, rapid progress in combining structural biology with other techniques is revealing the similarities and the differences in virion structure and function between ZIKV and related flaviviruses.

Keywords: cryo-EM, maturation, neutralizing antibodies, receptors, Zika virus
Jawahar Raina
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Generation of monoclonal antibodies against native viral proteins using antigen-expressing mammalian cells for mouse immunization

Generation of monoclonal antibodies against native viral proteins using antigen-expressing mammalian cells for mouse immunization

Abstract

Background

Due to their rising incidence and progressive geographical spread, infections with mosquito-borne viruses, such as dengue (DENV), chikungunya and zika virus, have developed into major public health challenges. Since all of these viruses may cause similar symptoms and can occur in concurrent epidemics, tools for their differential diagnosis and epidemiological monitoring are of urgent need.

Results

Here we report the application of a novel strategy to rapidly generate monoclonal antibodies (mAbs) against native viral antigens, exemplified for the DENV nonstructural glycoprotein 1 (NS1). The described system is based on the immunization of mice with transfected mammalian cells expressing the target antigens in multiple displays on their cell surface and thereby presenting them efficiently to the host immune system in their native conformation. By applying this cell-based approach to the DENV NS1 protein of serotypes 1 (D1NS1) and 4 (D4NS1), we were able to rapidly generate panels of DENV NS1 serotype cross-reactive, as well as D1NS1- and D4NS1 serotype-specific mAbs. Our data show that the generated mAbs were capable of recognizing the endogenous NS1 protein in DENV-containing biological samples.

Conclusion

The use of this novel immunization strategy, allows for a fast and efficient generation of hybridoma cell lines, producing mAbs against native viral antigens. Envisaged applications of the mAbs include the development of test platforms enabling a differentiation of the DENV serotypes and high resolution immunotyping for epidemiological studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12896-016-0314-5) contains supplementary material, which is available to authorized users.

Keywords: Dengue virus, NS1 protein, Mouse immunization, HEK cells, Transfection, Hybridoma technology, Monoclonal antibodies
Jawahar Raina
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Public antibodies to malaria antigens generated by two LAIR1 insertion modalities

Public antibodies to malaria antigens generated by two LAIR1 insertion modalities

Abstract

We previously described two donors in whom the extracellular domain of LAIR1, a collagen-binding inhibitory receptor encoded on chromosome 19, was inserted between the V and the DJ segments of an antibody. This insertion generated, through somatic mutations, broadly reactive antibodies against RIFINs, a type of variant antigen expressed on the surface of Plasmodium falciparum-infected erythrocytes (IEs). To investigate how frequently such antibodies are produced in response to malaria infection, we screened plasma from two large cohorts of individuals living in malaria-endemic regions. We report that 5-10% of malaria-exposed individuals, but none of the European blood donors tested, have high levels of LAIR1-containing antibodies that dominate the response to IEs without conferring enhanced protection against febrile malaria. By analyzing the antibody-producing B cell clones at the protein, cDNA and gDNA level, we characterized additional LAIR1 insertions between the V and DJ segments and discovered a second insertion modality whereby the LAIR1 exon encoding the extracellular domain and flanking intronic sequences are inserted into the switch region. By exon shuffling, this mechanism leads to the production of bispecific antibodies in which the LAIR1 domain is precisely positioned at the elbow between the VH and CH1 domains. Additionally, in one donor the gDNA encoding the VH and CH1 domains was deleted, leading to the production of a camel-like LAIR1-containing antibody. Sequencing of the switch regions of memory B cells from European blood donors revealed frequent templated inserts originating from transcribed genes that, in rare cases, comprised exons with orientation and frame compatible with expression. Collectively, these results reveal different modalities of LAIR1 insertion that lead to public and dominant antibodies against IEs and suggest that insertion of templated DNA represents an additional mechanism of antibody diversification that can be selected in the immune response against pathogens and exploited for B cell engineering.

Jawahar Raina
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Dengue Virus Induces Increased Activity of the Complement Alternative Pathway in Infected Cells

Dengue Virus Induces Increased Activity of the Complement Alternative Pathway in Infected Cells

ABSTRACT

Severe dengue virus (DENV) infection is associated with overactivity of the complement alternative pathway (AP) in patient studies. Here, the molecular changes in components of the AP during DENV infection in vitro were investigated. mRNA for factor H (FH), a major negative regulator of the AP, was significantly increased in DENV-infected endothelial cells (EC) and macrophages, but, in contrast, production of extracellular FH protein was not. This discord was not seen for the AP activator factor B (FB), with DENV induction of both FB mRNA and protein, nor was it seen with Toll-like receptor 3 or 4 stimulation of EC and macrophages, which induces both FH and FB mRNA and protein. Surface-bound and intracellular FH protein was, however, induced by DENV, but only in DENV antigen-positive cells, while in two other DENV-susceptible immortalized cell lines (ARPE-19 and human retinal endothelial cells), FH protein was induced both intracellularly and extracellularly by DENV infection. Regardless of the cell type, there was an imbalance in AP components and an increase in markers of complement AP activity associated with DENV-infected cells, with lower FH relative to FB protein, an increased ability to promote AP-mediated lytic activity, and increased deposition of complement component C3b on the surface of DENV-infected cells. For EC in particular, these changes are predicted to result in higher complement activity in the local cellular microenvironment, with the potential to induce functional changes that may result in increased vascular permeability, a hallmark of dengue disease.

IMPORTANCE Dengue virus (DENV) is a significant human viral pathogen with a global medical and economic impact. DENV may cause serious and life-threatening disease, with increased vascular permeability and plasma leakage. The pathogenic mechanisms underlying these features remain unclear; however, overactivity of the complement alternative pathway has been suggested to play a role. In this study, we investigate the molecular events that may be responsible for this observed alternative pathway overactivity and provide novel findings of changes in the complement system in response to DENV infection in primary cell types that are a major target for DENV infection (macrophages) and pathogenesis (endothelial cells) in vivo. Our results suggest a new dimension of cellular events that may influence endothelial cell barrier function during DENV infection that could expand strategies for developing therapeutics to prevent or control DENV-mediated vascular disease.

KEYWORDS: complement, dengue virus, factor Ha
Jawahar Raina
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155 results