IFIT1 Differentially Interferes with Translation and Replication of Alphavirus Genomes and Promotes Induction of Type I Interferon.

Alphaviruses are a group of widely distributed human and animal pathogens. It is well established that their replication is sensitive to type I IFN treatment, but the mechanism of IFN inhibitory function remains poorly understood. Using a new experimental system, we demonstrate that in the presence of IFN-ß, activation of interferon-stimulated genes (ISGs) does not interfere with either attachment of alphavirus virions to the cells, or their entry and nucleocapsid disassembly. However, it strongly affects translation of the virion-delivered virus-specific RNAs. One of the ISG products, IFIT1 protein, plays a major role in this translation block, although an IFIT1-independent mechanism is also involved. The 5'UTRs of the alphavirus genomes were found to differ significantly in their ability to drive translation in the presence of increased concentration of IFIT1. Prior studies have shown that adaptation of naturally circulating alphaviruses to replication in tissue culture results in accumulation of mutations in the 5'UTR, which increase the efficiency of the promoter located in the 5'end of the genome. Here, we show that these mutations also decrease resistance of viral RNA to IFIT1-induced translation inhibition. In the presence of higher levels of IFIT1, alphaviruses with wt 5'UTRs became potent inducers of type I IFN, suggesting a new mechanism of type I IFN induction. We applied this knowledge of IFIT1 interaction with alphaviruses to develop new attenuated variants of Venezuelan equine encephalitis and chikungunya viruses that are more sensitive to the antiviral effects of IFIT1, and thus could serve as novel vaccine candidates.

FungalRV: adhesin prediction and immunoinformatics portal for human fungal pathogens.

BACKGROUND: The availability of sequence data of human pathogenic fungi generates opportunities to develop Bioinformatics tools and resources for vaccine development towards benefitting at-risk patients. DESCRIPTION: We have developed a fungal adhesin predictor and an immunoinformatics database with predicted adhesins. Based on literature search and domain analysis, we prepared a positive dataset comprising adhesin protein sequences from human fungal pathogens Candida albicans, Candida glabrata, Aspergillus fumigatus, Coccidioides immitis, Coccidioides posadasii, Histoplasma capsulatum, Blastomyces dermatitidis, Pneumocystis carinii, Pneumocystis jirovecii and Paracoccidioides brasiliensis. The negative dataset consisted of proteins with high probability to function intracellularly. We have used 3945 compositional properties including frequencies of mono, doublet, triplet, and multiplets of amino acids and hydrophobic properties as input features of protein sequences to Support Vector Machine. Best classifiers were identified through an exhaustive search of 588 parameters and meeting the criteria of best Mathews Correlation Coefficient and lowest coefficient of variation among the 3 fold cross validation datasets. The "FungalRV adhesin predictor" was built on three models whose average Mathews Correlation Coefficient was in the range 0.89-0.90 and its coefficient of variation across three fold cross validation datasets in the range 1.2% - 2.74% at threshold score of 0. We obtained an overall MCC value of 0.8702 considering all 8 pathogens, namely, C. albicans, C. glabrata, A. fumigatus, B. dermatitidis, C. immitis, C. posadasii, H. capsulatum and P. brasiliensis thus showing high sensitivity and specificity at a threshold of 0.511. In case of P. brasiliensis the algorithm achieved a sensitivity of 66.67%. A total of 307 fungal adhesins and adhesin like proteins were predicted from the entire proteomes of eight human pathogenic fungal species. The immunoinformatics analysis data on these proteins were organized for easy user interface analysis. A Web interface was developed for analysis by users. The predicted adhesin sequences were processed through 18 immunoinformatics algorithms and these data have been organized into MySQL backend. A user friendly interface has been developed for experimental researchers for retrieving information from the database. CONCLUSION: FungalRV webserver facilitating the discovery process for novel human pathogenic fungal adhesin vaccine has been developed.

Immunoproteomic analysis of the protective response obtained from vaccination with Candida albicans ecm33 cell wall mutant in mice.

Systemic candidiasis remains a major cause of disease and death, particularly among immunocompromised patients. The cell wall of Candida albicans defines the interface between host and pathogen and surface proteins are major elicitors of host immune responses during candidiasis. The C. albicans ecm33 mutant (RML2U) presents an altered cell wall, which entails an increase in the outermost protein layer. Vaccination of BALB/c mice with RML2U mutant protected them from a subsequent lethal infection with virulent strain SC5314 in a systemic candidiasis model. Using immunoproteomics (2-DE followed by Immunoblotting) we detected 29 immunoreactive proteins specifically recognized by antibodies from vaccinated mice sera, six of which are described as immunogenic for the first time (Gnd1p, Cit1p, Rpl10Ep, Yst1p, Cys4p, Efb1p). Furthermore, identification of wild type and mutant cell surface proteome (surfome), confirmed us that the mutant surfome presented a larger number of proteins than the wild type. Interestingly, proteins exclusively identified in the mutant surfome (Met6p, Eft2p, Tkl1p, Rpl10Ep, Atp1p, Atp2p) were also detected as immunogenic, supporting the idea that their surface location enhances their immunoprotective capacity.

Experimental animal models of coccidioidomycosis.

Experimental models of coccidioidomycosis performed using various laboratory animals have been, and remain, a critical component of elucidation and understanding of the pathogenesis and host resistance to infection with Coccidioides spp., as well as to development of more efficacious antifungal therapies. The general availability of genetically defined strains, immunological reagents, ease of handling, and costs all contribute to the use of mice as the primary laboratory animal species for models of this disease. Five types of murine models are studied and include primary pulmonary disease, intraperitoneal with dissemination, intravenous infection emulating systemic disease, and intracranial or intrathecal infection emulating meningeal disease. Each of these models has been used to examine various aspects of host resistance, pathogenesis, or antifungal therapy. Other rodent species, such as rat, have been used much less frequently. A rabbit model of meningeal disease, established by intracisternal infection, has proven to model human meningitis well. This model is useful in studies of host response, as well as in therapy studies. A variety of other animal species including dogs, primates, and guinea pigs have been used to study host response and vaccine efficacy. However, cost and increased needs of animal care and husbandry are limitations that influence the use of the larger animal species.

Characteristics of the protective subcellular coccidioidal T27K vaccine.

While the whole killed spherule vaccine, protective in mice and monkeys, did not prevent coccidioidal disease in humans, the 27K vaccine, a soluble derivative, retains protective activity in mice with little irritant action. Gel filtration and anion exchange fractions of thimerosal-inactivated spherules (T27K), when administered with alum adjuvant, also protect mice against lethal respiratory coccidioidal challenge. However, the superb protection afforded by T27K antigens is maintained for some 3 months, but may then diminish. This appears unrelated to the aging of the mice. Prolongation of the protective action may require addition of a different adjuvant or administration of booster doses of vaccine.

A proteomic-based approach for the identification of Candida albicans protein components present in a subunit vaccine that protects against disseminated candidiasis.

Candidiasis has become a prevalent infection in different types of immunocompromised patients. The cell wall of Candida albicans plays important functions during the host-fungus interactions. Cell wall (surface) proteins of C. albicans are major elicitors of host immune responses during candidiasis, and represent candidates for vaccine development. Groups of mice were vaccinated subcutaneously with a beta-mercaptoethanol (beta-ME) extract from C. albicans containing cell wall proteins. Vaccinated mice were then infected with a lethal dose of C. albicans. Increased survival and decreased fungal burden were observed in vaccinated mice as compared to a control group, and 75% of vaccinated mice with the beta-ME extract survived this otherwise lethal infection. We used a proteomic approach (2-DE followed by immunoblotting) to demonstrate a complex polypeptidic pattern associated with the beta-ME extract used in the vaccine formulation and to detect immunogenic components recognized by antibodies in immune sera from vaccinated animals. Reactive protein spots were identified by MALDI-TOF-MS and searches in genomic databases. As a conclusion, vaccination strategies using C. albicans cell wall proteins induce protective responses. These antigens can be identified by proteomic approaches and may be used as components of subcellular vaccines against candidiasis.

Antibodies to keyhole limpet hemocyanin cross-react with an epitope on the polysaccharide capsule of Cryptococcus neoformans and other carbohydrates: implications for vaccine development.

Cryptococcus neoformans causes a life-threatening meningoencephalitis in AIDS patients. Mice immunized with a glycoconjugate vaccine composed of the glucuronoxylomannan (GXM) component of the cryptococcal capsular polysaccharide conjugated to tetanus toxoid produce Abs that can be either protective or nonprotective. Because nonprotective Abs block the efficacy of protective Abs, an effective vaccine must focus the Ab response on a protective epitope. Mice immunized with peptide mimetics of GXM conjugated to keyhole limpet hemocyanin (KLH) with glutaraldehyde developed Abs to GXM. However, control peptides P315 and P24 conjugated to KLH also elicited Abs to GXM. GXM-binding Abs from mice immunized with P315-KLH were inhibited by KLH treated with glutaraldehyde (KLH-g), but not by P315. Furthermore, KLH-g inhibited binding of GXM by serum of mice immunized with GXM-TT, indicating that glutaraldehyde treatment of KLH reveals an epitope(s) that cross-reacts with GXM. Vaccination with KLH-g or unmodified KLH elicited Abs to GXM, but did not confer protection against C. neoformans, suggesting the cross-reactive epitope on KLH was not protective. This was supported by the finding that 4H3, a nonprotective mAb, cross-reacted strongly with KLH-g. Sera from mice immunized with either native KLH or KLH-g cross-reacted with several other carbohydrate Ags, many of which have been conjugated to KLH for vaccine development. This study illustrates how mAbs can be used to determine the efficacy of potential vaccines, in addition to describing the complexity of using KLH and glutaraldehyde in the development of vaccines to carbohydrate Ags.

High affinity mimotope of the polysaccharide capsule of Cryptococcus neoformans identified from an evolutionary phage peptide library.

Cryptococcus neoformans causes a life-threatening meningoencephalitis in a significant percentage of AIDS patients. Mice immunized with a glycoconjugate vaccine composed of the glucuronoxylomannan (GXM) component of the cryptococcal capsular polysaccharide conjugated to tetanus toxoid (TT) produce Abs that, based on the epitope recognized, can be either protective or nonprotective. Since nonprotective Abs block the efficacy of protective Abs, we are interested in developing a vaccine that would focus the immune response specifically to protective epitopes. Previously, we screened a phage display library with 2H1, a protective anti-GXM mAb, and isolated PA1, a representative peptide that had a K(d) of 295 nM for 2H1. Mice immunized with PA1 conjugated to keyhole limpet hemocyanin developed high anti-peptide (1/13,000), but low anti-GXM (maximum, 1/200) titers. We now report our efforts to improve this vaccine by screening a sublibrary with six random amino acids added to either end of the PA1 motif to identify higher affinity peptides. P206.1, a peptide isolated from this sublibrary, had 80-fold higher affinity for 2H1 (K(d) = 3.7 nM) than PA1. P206.1 bound protective, but not nonprotective, anti-GXM Abs. Mice immunized with P206.1 conjugated to various carriers did not mount an Ab response to GXM despite developing high anti-peptide titers. However, mice primed with GXM-TT and boosted with P206.1-TT developed significant anti-GXM titers (maximum, 1/180,000). This latter immunization scheme focused the immune response on protective epitopes, since only 2-5% of these titers were directed against nonprotective de-O-acetylated GXM epitopes compared with 20-60% in animals primed and boosted with GXM-TT.