Single dose of glycoprotein K (gK)-deleted HSV-1 live-attenuated virus protects mice against lethal vaginal challenge with HSV-1 and HSV-2 and induces lasting T cell memory immune responses.

BACKGROUND: Herpes simplex virus type-1(HSV-1) and HSV-2 are important human pathogens that cause significant ocular and urogenital complications, respectively. We have previously shown that HSV-1 virions lacking glycoprotein K (gK) are unable to enter into neurons via synaptic axonal membranes and be transported in either retrograde or anterograde manner. Here, we tested the ability of HSV-1 (F) gK-null to protect against lethal challenge with either highly virulent ocular HSV-1 (McKrae strain), or genital HSV-2 (G strain). The gK-null virus vaccine efficiently protected mice against lethal vaginal infection with either HSV-1(McKrae) or HSV-2 (G). RESULTS: Female mice were immunized via a single intramuscular injection with 106 PFU of the gK-null virus. Immunized mice were treated with Depo-Provera fourteen days after vaccination and were challenged via the vaginal route one week later. Ninety percent of mice vaccinated with the gK-null virus survived HSV-1 (McKrae) challenge, while 70% of these mice survived after HSV-2 (G) challenge. Moreover, all vaccinated mice exhibited substantially reduced disease symptoms irrespective of HSV-1 or HSV-2 challenge as compared to the mock vaccinated challenge group. T-cell memory immune responses to specific glycoprotein B (gB) and glycoprotein D (gD) peptide epitopes were detectable at 7 months post vaccination. CONCLUSIONS: These results suggest that the highly attenuated, non-neurotropic gK-null virus may be used as an effective vaccine to protect against both virulent HSV-1 and HSV-2 genital infections and induce lasting immune responses.

The herpes simplex virus type 1 UL20 protein and the amino terminus of glycoprotein K (gK) physically interact with gB.

Herpes simplex virus type 1 (HSV-1) glycoprotein K (gK) and the UL20 protein (UL20p) are strictly required for virus-induced cell fusion, and mutations within either the gK or UL20 gene cause extensive cell fusion (syncytium formation). We have shown that gK forms a functional protein complex with UL20p, which is required for all gK and UL20p-associated functions in the HSV-1 life cycle. Recently, we showed that the amino-terminal 82 amino acids (aa) of gK (gKa) were required for the expression of the syncytial phenotype of the mutant virus gBDelta28 lacking the carboxyl-terminal 28 amino acids of gB (V. N. Chouljenko, A. V. Iyer, S. Chowdhury, D. V. Chouljenko, and K. G. Kousoulas, J. Virol. 83:12301-12313, 2009). This work suggested that the amino terminus of gK may directly or indirectly interact with gB and/or other viral glycoproteins. Two-way coimmunoprecipitation experiments revealed that UL20p interacted with gB in infected cells. Furthermore, the gKa peptide was coimmunoprecipitated with gB but not gD. Three recombinant baculoviruses were constructed, expressing the amino-terminal 82 aa of gKa together with either the extracellular portion of gB (30 to 748 aa), gD (1 to 340 aa), or gH (1 to 792 aa), respectively. Coimmunoprecipitation experiments revealed that gKa physically interacted with the extracellular portions of gB and gH but not gD. Three additional recombinant baculoviruses expressing gKa and truncated gBs encompassing aa 30 to 154, 30 to 364, and 30 to 500 were constructed. Coimmunoprecipitation experiments showed that gKa physically interacted with all three truncated gBs. Computer-assisted prediction of possible gKa binding sites on gB suggested that gKa may interact predominantly with gB domain I (E. E. Heldwein, H. Lou, F. C. Bender, G. H. Cohen, R. J. Eisenberg, and S. C. Harrison, Science 313:217-220, 2006). These results imply that the gK/UL20p protein complex modulates the fusogenic properties of gB and gH via direct physical interactions.

The amino terminus of herpes simplex virus type 1 glycoprotein K (gK) modulates gB-mediated virus-induced cell fusion and virion egress.

Herpes simplex virus type 1 (HSV-1)-induced cell fusion is mediated by viral glycoproteins and other membrane proteins expressed on infected cell surfaces. Certain mutations in the carboxyl terminus of HSV-1 glycoprotein B (gB) and in the amino terminus of gK cause extensive virus-induced cell fusion. Although gB is known to be a fusogenic glycoprotein, the mechanism by which gK is involved in virus-induced cell fusion remains elusive. To delineate the amino-terminal domains of gK involved in virus-induced cell fusion, the recombinant viruses gKDelta31-47, gKDelta31-68, and gKDelta31-117, expressing gK carrying in-frame deletions spanning the amino terminus of gK immediately after the gK signal sequence (amino acids [aa] 1 to 30), were constructed. Mutant viruses gKDelta31-47 and gKDelta31-117 exhibited a gK-null (DeltagK) phenotype characterized by the formation of very small viral plaques and up to a 2-log reduction in the production of infectious virus in comparison to that for the parental HSV-1(F) wild-type virus. The gKDelta31-68 mutant virus formed substantially larger plaques and produced 1-log-higher titers than the gKDelta31-47 and gKDelta31-117 mutant virions at low multiplicities of infection. Deletion of 28 aa from the carboxyl terminus of gB (gBDelta28syn) caused extensive virus-induced cell fusion. However, the gBDelta28syn mutation was unable to cause virus-induced cell fusion in the presence of the gKDelta31-68 mutation. Transient expression of a peptide composed of the amino-terminal 82 aa of gK (gKa) produced a glycosylated peptide that was efficiently expressed on cell surfaces only after infection with the HSV-1(F), gKDelta31-68, DeltagK, or UL20-null virus. The gKa peptide complemented the gKDelta31-47 and gKDelta31-68 mutant viruses for infectious-virus production and for gKDelta31-68/gBDelta28syn-mediated cell fusion. These data show that the amino terminus of gK modulates gB-mediated virus-induced cell fusion and virion egress.

Clostridium taeniosporum is a close relative of the Clostridium botulinum Group II.

Clostridium taeniosporum is a Gram-positive, anaerobic, rod-shaped non-toxigenic organism isolated from Crimean lake silt. It is unique in forming spores from which about twelve large, flat, ribbon-like appendages emanate. These ribbon-like structures, about 4.5 microm long and 0.45 microm wide, are assembled from smaller fibrils with 5 nm diameter spherical heads attached to thin tails about 1-2 nm in diameter and about 40 nm in length. The appendages have four major components, a glycoprotein with a collagen-like region, two proteins each of which contains two conserved domains of unknown function, and an ortholog of the Bacillus subtilis spore morphogenetic protein SpoVM. Genes for three of these and other, possibly related proteins, cluster on two chromosome fragments. Here we report that C. taeniosporum is saccharolytic, non-proteolytic, and produces both acetic and butyric acid fermentation products. It synthesizes alpha-D-glucosidase and N-acetyl-beta,D-glucoseaminidase constitutively. These physiological properties are similar to those of the C. botulinum Group II. Genotypically, C. taeniosporum is also closely related to the same Group II, based on 16S rDNA sequences. C. taeniosporum differs from typical C. botulinum Group II strains because it is non-toxigenic and in forming the ribbon-like spore appendages. These major differences among otherwise closely related organisms suggest lateral transfer of genes for appendage synthesis and for toxigenicity.

Oral administration of a Spirulina extract enriched for Braun-type lipoproteins protects mice against influenza A (H1N1) virus infection.

A growing body of research indicates that oral administration of bacteria (such as probiotics) can exhibit a protective effect against influenza A (H1N1) viral infection in mice. In the present study, we used a mouse model to examine whether oral administration of Immulina(®), a commercial extract from the cyanobacteria Arthrospira (Spirulina) platensis, can reduce the severity of illness resulting from influenza A (H1N1) viral infection. The main active compounds within Immulina(®) are bacterial Braun-type lipoproteins that activate innate immune cells through a toll-like receptor (TLR) 2-dependent pathway. Mice that were fed Immulina(®) for 30 days before and 21 days after infection with influenza A (H1N1) virus exhibited a statistically significant reduction in the severity of infection. Compared to the control group, Immulina(®)-fed mice exhibited less weight loss, increased appetite, decreased clinical signs of disease, and lower lung histopathology scores. The results from the present study adds to the increasing evidence that oral administration of bacterial components that activate innate immune cells, whether derived from a bacterial preparation (probiotics or cyanobacteria) or from plant material containing endophytic bacteria, can exhibit a protective effect against influenza A (H1N1) viral infection.

A review of vaccine approaches for West Nile virus.

The West Nile virus (WNC) first appeared in North America in 1999. The North American lineages of WNV were characterized by the presence of neuroinvasive and neurovirulent strains causing disease and death in humans, birds and horses. The 2012 WNV season in the United States saw a massive spike in the number of neuroinvasive cases and deaths similar to what was seen in the 2002-2003 season, according to the West Nile virus disease cases and deaths reported to the CDC by year and clinical presentation, 1999-2012, by ArboNET (Arboviral Diseases Branch, Centers for Disease Control and Prevention). In addition, the establishment and recent spread of lineage II WNV virus strains into Western Europe and the presence of neurovirulent and neuroinvasive strains among them is a cause of major concern. This review discusses the advances in the development of vaccines and biologicals to combat human and veterinary West Nile disease.

Complete genome analysis and virulence characteristics of the Louisiana West Nile virus strain LSU-AR01.

West Nile virus (WNV) is a member of the Flaviriridae family, which can cause significant morbidity and mortality in birds, horses, and humans. The WNV-LSU-AR01 strain was isolated from a dead blue jay in Louisiana in 2001. Phylogenetic analysis using 75 full WNV genomes revealed that the LSU-AR01 strain belongs to a distinct subclade among the North American strains. The LSU-AR01 strain differed from the NY-99 prototypic strain by 26 nucleotides causing six amino acid changes. An asparagine-to-lysine change was located immediately proximal to a known CD8(+)T cell epitope in NS4B, while a glutamine-to-lysine change was located within a predicted CD8(+)T cell epitope in NS5. The LSU-AR01 strain caused pronounced neuronal necrosis, perivascular cuffing and gliosis in comparison to the NY-99-infected mice. These results suggest that the previously identified Connecticut strains may contain highly neurovirulent strains such as the LSU-AR01 that have spread in North America.

Recombinant vesicular stomatitis virus-based west Nile vaccine elicits strong humoral and cellular immune responses and protects mice against lethal challenge with the virulent west Nile virus strain LSU-AR01.

Vesicular stomatitis virus (VSV) has been extensively utilized as a viral vector system for the induction of protective immune responses against a variety of pathogens. We constructed recombinant VSVs specifying either the Indiana or Chandipura virus G glycoprotein and expressing the West Nile virus (WNV) envelope (E) glycoprotein. Mice were intranasally vaccinated using a prime (Indiana)-boost (Chandipura) immunization approach and challenged with the virulent WNV-LSU-AR01. Ninety-percent (9 of 10) of the vaccinated mice survived as compared to 10% of the mock-vaccinated mice after WNV lethal challenge. Histopathological examination of brain tissues revealed neuronal necrosis in mock-vaccinated mice but not in vaccinated mice, and vaccinated, but not mock-vaccinated mice developed a strong neutralizing antibody response against WNV. Extensive immunological analysis using polychromatic flow cytometry staining revealed that vaccinated, but not mock-vaccinated mice developed robust cellular immune responses as evidenced by up-regulation of CD4(+) CD154(+) IFNgamma(+) T cells in vaccinated, but not mock-vaccinated mice. Similarly, vaccinated mice developed robust E-glycoprotein-specific CD8(+) T cell immune responses as evidenced by the presence of a high percentage of CD8(+) CD62L(low) IFNgamma(+) cells. In addition, a sizeable population of CD8(+) CD69(+) cells was detected indicating E-specific activation of mature T cells and CD4(+) CD25(+) CD127(low) T regulatory (T reg) cells were down-regulated. These results suggest that VSV-vectored vaccines administered intranasally can efficiently induce protective humoral and cellular immune responses against WNV infections.