Newcastle disease virus expressing a dendritic cell-targeted HIV Gag protein induces a potent Gag-specific immune response in mice

Viral vaccine vectors have emerged as an attractive strategy for the development of a human immunodeficiency virus (HIV) vaccine. Recombinant Newcastle disease virus (rNDV) stands out as a vaccine vector since it has a proven safety profile in humans, it is a potent inducer of both alpha interferon (IFN-α) and IFN-β) production, and it is a potent inducer of dendritic cell (DC) maturation. Our group has previously generated an rNDV vector expressing a codon-optimized HIV Gag protein and demonstrated its ability to induce a Gag-specific CD8+ T cell response in mice. In this report we demonstrate that the Gag-specific immune response can be further enhanced by the targeting of the rNDV-encoded HIV Gag antigen to DCs. Targeting of the HIV Gag antigen was achieved by the addition of a single-chain Fv (scFv) antibody specific for the DC-restricted antigen uptake receptor DEC205 such that the DEC205 scFv-Gag molecule was encoded for expression as a fusion protein. The vaccination of mice with rNDV coding for the DC-targeted Gag antigen induced an enhanced Gag-specific CD8+ T cell response and enhanced numbers of CD4+ T cells and CD8+ T cells in the spleen relative to vaccination with rNDV coding for a nontargeted Gag antigen. Importantly, mice vaccinated with the DEC205-targeted vaccine were better protected from challenge with a recombinant vaccinia virus expressing the HIV Gag protein. Here we demonstrate that the targeting of the HIV Gag antigen to DCs via the DEC205 receptor enhances the ability of an rNDV vector to induce a potent antigen-specific immune response

Influenza Virus Vaccine Based on the Conserved Hemagglutinin Stalk Domain

Although highly effective in the general population when well matched to circulating influenza virus strains, current influenza vaccines are limited in their utility due to the narrow breadth of protection they provide. The strain specificity of vaccines presently in use mirrors the exquisite specificity of the neutralizing antibodies that they induce, that is, antibodies which bind to the highly variable globular head domain of hemagglutinin (HA). Herein, we describe the construction of a novel immunogen comprising the conserved influenza HA stalk domain and lacking the globular head. Vaccination of mice with this headless HA construct elicited immune sera with broader reactivity than those obtained from mice immunized with a full-length HA. Furthermore, the headless HA vaccine provided full protection against death and partial protection against disease following lethal viral challenge. Our results suggest that the response induced by headless HA vaccines is sufficiently potent to warrant their further development toward a universal influenza virus vaccine

Evaluation of serological cross-reactivity and cross-neutralization between the United States porcine epidemic diarrhea virus prototype and S-INDEL-variant strains

BACKGROUND: At least two genetically different porcine epidemic diarrhea virus (PEDV) strains have been identified in the United States (U.S. PEDV prototype and S-INDEL-variant strains). The current serological assays offered at veterinary diagnostic laboratories for detection of PEDV-specific antibody are based on the U.S. PEDV prototype strain. The objectives of this study were: 1) isolate the U.S. PEDV S-INDEL-variant strain in cell culture; 2) generate antisera against the U.S. PEDV prototype and S-INDEL-variant strains by experimentally infecting weaned pigs; 3) determine if the various PEDV serological assays could detect antibodies against the U.S. PEDV S-INDEL-variant strain and vice versa. RESULTS: A U.S. PEDV S-INDEL-variant strain was isolated in cell culture in this study. Three groups of PEDV-negative, 3-week-old pigs (five pigs per group) were inoculated orally with a U.S. PEDV prototype isolate (previously isolated in our lab), an S-INDEL-variant isolate or virus-negative culture medium. Serum samples collected at 0, 7, 14, 21 and 28 days post inoculation were evaluated by the following PEDV serological assays: 1) indirect fluorescent antibody (IFA) assays using the prototype and S-INDEL-variant strains as indicator viruses; 2) virus neutralization (VN) tests against the prototype and S-INDEL-variant viruses; 3) PEDV prototype strain whole virus based ELISA; 4) PEDV prototype strain S1-based ELISA; and 5) PEDV S-INDEL-variant strain S1-based ELISA. The positive antisera against the prototype strain reacted to and neutralized both prototype and S-INDEL-variant viruses, and the positive antisera against the S-INDEL-variant strain also reacted to and neutralized both prototype and S-INDEL-variant viruses, as examined by IFA antibody assays and VN tests. Antibodies against the two PEDV strains could be detected by all three ELISAs although detection rates varied to some degree. CONCLUSIONS: These data indicate that the antibodies against U.S. PEDV prototype and S-INDEL-variant strains cross-reacted and cross-neutralized both strains in vitro. The current serological assays based on U.S. PEDV prototype strain can detect antibodies against both U.S. PEDV strains

Widespread Occurrence of Dosage Compensation in Candida albicans

The important human pathogen Candida albicans possesses an unusual form of gene regulation, in which the copy number of an entire specific chromosome or a large portion of a specific chromosome changes in response to a specific adverse environment, thus, insuring survival. In the absence of the adverse environment, the altered portion of the genome can be restored to its normal condition. One major question is how C. albicans copes with gene imbalance arising by transitory aneuploid states. Here, we compared transcriptomes from cells with either two copies or one copy of chromosome 5 (Ch5) in, respectively, a diploid strain 3153A and its representative derivative Sor55. Statistical analyses revealed that at least 40% of transcripts from the monosomic Ch5 are fully compensated to a disomic level, thus, indicating the existence of a genome-wide mechanism maintaining cellular homeostasis. Only approximately 15% of transcripts were diminished twofold in accordance with what would be expected for Ch5 monosomy. Another minor portion of approximately 6% of transcripts, unexpectedly, increased up to twofold and higher than the disomic level, demonstrating indirect control by monosomy. Array comparative genome hybridization revealed that only few out of approximately 500 genes on the monosomic Ch5b were duplicated, thus, not causing a global up regulation. Dosage compensation was confirmed with several representative genes from another monosomic Ch5a in the mutant Sor60. We suggest that C. albicans's unusual regulation of gene expression by the loss and gain of entire chromosomes is coupled with widespread compensation of gene dosage at the transcriptional level

Rewiring the RNAs of influenza virus to prevent reassortment

Influenza viruses contain segmented, negative-strand RNA genomes. Genome segmentation facilitates reassortment between different influenza virus strains infecting the same cell. This phenomenon results in the rapid exchange of RNA segments. In this study, we have developed a method to prevent the free reassortment of influenza A virus RNAs by rewiring their packaging signals. Specific packaging signals for individual influenza virus RNA segments are located in the 5′ and 3′ noncoding regions as well as in the terminal regions of the ORF of an RNA segment. By putting the nonstructural protein (NS)-specific packaging sequences onto the ORF of the hemagglutinin (HA) gene and mutating the packaging regions in the ORF of the HA, we created a chimeric HA segment with the packaging identity of an NS gene. By the same strategy, we made an NS gene with the packaging identity of an HA segment. This rewired virus had the packaging signals for all eight influenza virus RNAs, but it lost the ability to independently reassort its HA or NS gene. A similar approach can be applied to the other influenza A virus segments to diminish their ability to form reassortant viruses

Expression of Transgenes from Newcastle Disease Virus with a Segmented Genome▿

Paramyxoviruses belong to the Paramyxoviridae family of the order Mononegavirales. They have a nonsegmented negative-stranded RNA genome and can cause a number of diseases in humans and animals. We generated a recombinant Newcastle disease virus (NDV) possessing a two-segmented genome. Each genomic segment is flanked by authentic NDV 3′ and 5′ noncoding termini allowing for efficient replication and transcription. A reporter gene encoding green fluorescent protein (GFP) was inserted into one segment, and a red fluorescent protein dsRed gene was inserted into the other segment in order to easily detect the replication and transcription of segments in infected cells. The rescued viruses grew well and were stable in embryonated chicken eggs over multiple passages. We were able to detect the expression of both reporter genes in the same cell infected with the virus possessing a segmented genome, and viral particles can contain either one or two types of RNA segments. We also rescued a two-segmented virus expressing GFP and the severe acute respiratory syndrome-associated coronavirus spike S protein, which is about 200 kDa. The chimeric virus extends the coding capacity of NDV by 30%, suggesting that the two-segmented NDV can be used for development of vaccines or gene therapy vectors carrying long and multiple transgenes

A Seven-Segmented Influenza A Virus Expressing the Influenza C Virus Glycoprotein HEF▿

Influenza viruses are classified into three types: A, B, and C. The genomes of A- and B-type influenza viruses consist of eight RNA segments, whereas influenza C viruses only have seven RNAs. Both A and B influenza viruses contain two major surface glycoproteins: the hemagglutinin (HA) and the neuraminidase (NA). Influenza C viruses have only one major surface glycoprotein, HEF (hemagglutinin-esterase fusion). By using reverse genetics, we generated two seven-segmented chimeric influenza viruses. Each possesses six RNA segments from influenza virus A/Puerto Rico/8/34 (PB2, PB1, PA, NP, M, and NS); the seventh RNA segment encodes either the influenza virus C/Johannesburg/1/66 HEF full-length protein or a chimeric protein HEF-Ecto, which consists of the HEF ectodomain and the HA transmembrane and cytoplasmic regions. To facilitate packaging of the heterologous segment, both the HEF and HEF-Ecto coding regions are flanked by HA packaging sequences. When introduced as an eighth segment with the NA packaging sequences, both viruses are able to stably express a green fluorescent protein (GFP) gene, indicating a potential use for these viruses as vaccine vectors to carry foreign antigens. Finally, we show that incorporation of a GFP RNA segment enhances the growth of seven-segmented viruses, indicating that efficient influenza A viral RNA packaging requires the presence of eight RNA segments. These results support a selective mechanism of viral RNA recruitment to the budding site

Colocalization of different influenza viral RNA segments in the cytoplasm before viral budding as shown by single-molecule sensitivity FISH analysis.

The Influenza A virus genome consists of eight negative sense, single-stranded RNA segments. Although it has been established that most virus particles contain a single copy of each of the eight viral RNAs, the packaging selection mechanism remains poorly understood. Influenza viral RNAs are synthesized in the nucleus, exported into the cytoplasm and travel to the plasma membrane where viral budding and genome packaging occurs. Due to the difficulties in analyzing associated vRNPs while preserving information about their positions within the cell, it has remained unclear how and where during cellular trafficking the viral RNAs of different segments encounter each other. Using a multicolor single-molecule sensitivity fluorescence in situ hybridization (smFISH) approach, we have quantitatively monitored the colocalization of pairs of influenza viral RNAs in infected cells. We found that upon infection, the viral RNAs from the incoming particles travel together until they reach the nucleus. The viral RNAs were then detected in distinct locations in the nucleus; they are then exported individually and initially remain separated in the cytoplasm. At later time points, the different viral RNA segments gather together in the cytoplasm in a microtubule independent manner. Viral RNAs of different identities colocalize at a high frequency when they are associated with Rab11 positive vesicles, suggesting that Rab11 positive organelles may facilitate the association of different viral RNAs. Using engineered influenza viruses lacking the expression of HA or M2 protein, we showed that these viral proteins are not essential for the colocalization of two different viral RNAs in the cytoplasm. In sum, our smFISH results reveal that the viral RNAs travel together in the cytoplasm before their arrival at the plasma membrane budding sites. This newly characterized step of the genome packaging process demonstrates the precise spatiotemporal regulation of the infection cycle