Abolghasem Baghian

CD8+ Cytotoxic T Lymphocyte Responses to Lytic Proteins of Human Herpes Virus 8 in Human Immunodeficiency Virus Type 1—Infected and —Uninfected Individuals

T cell immunity to lytic proteins of herpesviruses is important in host control of infection. We have characterized the cytotoxic T lymphocyte (CTL) response to 5 human herpesvirus 8 (HHV-8) homologues of lytic proteins in HHV-8-seropositive individuals. HLA class I-restricted, CD8(+) CTL responses to >/=1 HHV-8 lytic protein were detected in all 14 HHV-8-seropositive study subjects tested, with or without human immunodeficiency virus type 1 (HIV-1) infection, but not in any of 5 HHV-8-seronegative individuals. Seven of these study subjects with both HHV-8 and HIV-1 infection had greater anti-CTL reactivity to glycoprotein H (open-reading frame 22) than did the 7 study subjects infected only with HHV-8. Moreover, there was a strong, inverse correlation between HIV-1 load and glycoprotein H-specific CTL lysis in the study subjects infected with both viruses. CTL reactivity to HHV-8 lytic proteins may be involved in host control of HHV-8-related diseases, such as Kaposis sarcoma.

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

Abstract 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+ IFNγ+ 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+ CD62Llow IFNγ+ cells. In addition, a sizeable population of CD8+ CD69+ cells was detected indicating E-specific activation of mature T cells and CD4+ CD25+ CD127low 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.

The Herpes Simplex Virus Type 1 (HSV-1) Glycoprotein K(gK) is Essential for Viral Corneal Spread and Neuroinvasiveness

Purpose: To determine the role of herpes simplex virus-1 (HSV-1) glycoprotein K(gK) in corneal infection, neuroinvasion, and virus latency in trigeminal ganglia of mice. Methods: The recombinant virus HSV-1 (McKrae) Δ gK (MKΔ gK) carrying a deletion of the gK gene was constructed by insertional/deletion mutagenesis and replaced by a gene cassette constitutively expressing the enhanced green fluorescence protein. The gK deletion of the MKΔ gK virus was rescued to produce the wild-type-like virus MKgK. Balb/c mice were infected ocularly with either virus, and the infection pattern in the eye, clinical disease progression, and establishment of viral latency was monitored. Results: Mice infected with the MKΔ gK strain produced in a gK complementing cell line did not exhibit clinical signs when compared with mice infected with the MKgK virus. Direct visualization of infected eyes revealed that the MKΔ gK virus was unable to spread in mouse corneas, while the MKgK rescued virus spread efficiently. Nineteen of 20 scarified and 5/12 unscarified mice infected with the MKgK virus produced infectious virus after coculture with permissive cells, while 0/20 scarified and 0/12 unscarified mice infected with the MKΔ gK virus produced infectious virus. HSV DNA was detected in trigeminal ganglia by PCR in 19/20 scarified and 9/12 unscarified mice inoculated with MKgK, while HSV DNA was detected in the trigeminal ganglia of 3/20 scarified and 0/12 unscarified mice inoculated with MKΔ gK. Conclusions: The results show that HSV-1 gK is essential for efficient replication and spread in the corneal epithelium and trigeminal ganglia neuroinvasion in MKΔ gK inoculated mice.

Herpes simplex virus type-1(HSV-1) oncolytic and highly fusogenic mutants carrying the NV1020 genomic deletion effectively inhibit primary and metastatic tumors in mice

BackgroundThe NV1020 oncolytic herpes simplex virus type-1 has shown significant promise for the treatment of many different types of tumors in experimental animal models and human trials. Previously, we described the construction and use of the NV1020-like virus OncSyn to treat human breast tumors implanted in nude mice. The syncytial mutation gKsyn1 (Ala-to-Val at position 40) was introduced into the OncSyn viral genome cloned into a bacterial artificial chromosome using double-red mutagenesis in E. coli to produce the OncdSyn virus carrying syncytial mutations in both gB(syn3) and gK(syn1).ResultsThe OncdSyn virus caused extensive virus-induced cell fusion in cell culture. The oncolytic potential of the OncSyn and OncdSyn viruses was tested in the highly metastatic syngeneic mouse model system, which utilizes 4T1 murine mammary cancer cells implanted within the interscapular region of Balb/c mice. Mice were given three consecutive intratumor injections of OncSyn, OncdSyn, or phosphate buffered saline four days apart. Both OncSyn and OncdSyn virus injections resulted in significant reduction of tumor sizes (p < 0.05) compared to control tumors. Virus treated mice but not controls showed a marked reduction of metastatic foci in lungs and internal organs. Mouse weights were not significantly impacted by any treatment during the course of the entire study (p = 0.296).ConclusionThese results show that the attenuated, but highly fusogenic OncSyn and OncdSyn viruses can effectively reduce primary and metastatic breast tumors in immuncompetent mice. The available bac-cloned OncSyn and OncdSyn viral genomes can be rapidly modified to express a number of different anti-tumor and immunomodulatory genes that can further enhance their anti-tumor potency.

Protective immunity against lethal HSV-1 challenge in mice by nucleic acid-based immunisation with herpes simplex virus type-1 genes specifying glycoproteins gB and gD

DNA-based vaccines were employed to assess protective immunity against herpes simplex virus in experimental infections of hairless (strain SKH1) and BALB/c mice. Mice were vaccinated with plasmids containing the herpes simplex virus type-1 (HSV-1) glycoprotein B (gB) or D (gD) genes under the human cytomegalovirus immediate-early promoter control. Vaccines were injected intramuscularly (i.m.) or intraperitoneally (i.p.) as purified DNA alone or as formulations supplemented with different non-ionic block copolymers. Antibody responses were assessed by immunofluorescence and radio-immunoprecipitation assays. Mice inoculated with either gB or gD plasmid, alone or with non-ionic block copolymers CRL 1029 and CRL 1190, produced high levels of antibodies specific for gB or gD. Three weeks after the last vaccination, mice were challenged with a clinical HSV-1 isolate (ABGK-1) by inoculation of a shaved and subsequently scarified area between the third and fourth lumbar vertebrae. Mice immunised with either gD or gB plasmid alone or mixed with copolymers were protected against lethal HSV-1 challenge when immunisation was performed via the i.m. route. Immunisations given via the i.p. route induced humoral responses in some mice and protected the animals against lethal HSV-1 challenge only when the formulations contained copolymers. The BALB/c mouse model was shown to be as good a model as the hairless mouse model.

Production of a Rabbit Anti-Cockatiel Immunoglobulin G and Characterization of Its Cross-Reactivities with Immunoglobulin G of Other Psittacine Species

The purpose of this work was to produce rabbit anti-cockatiel immunoglobulin G (IgG) and compare its cross-reactivity with sera from eight other psittacine birds: Quaker parakeet, budgerigar, green-wing macaw, blue-fronted Amazon parrot, eclectus parrot, African grey parrot, Patagonian conure, Moluccan cockatoo. Cockatiel IgG did not bind to protein A or G; therefore, these proteins could not be used in column chromatography to isolate the IgG. A combination of serum IgG precipitation by ammonium sulfate and yolk IgG extraction from egg was loaded in sodium dodecyl sulfate-polyacrylamide gel upon which the IgG was resolved by electrophoresis. The resolved IgG in sodium dodecyl sulfate-polyacrylamide gel was stained with Coomassie blue, cut, crushed in phosphate-buffered saline, and injected into rabbits. The rabbit anti-cockatiel IgG produced in this way reacted with a single protein in gel immunodiffusion assay with all nine psittacine bird sera but not with those of chicken and ostrich. Immunoelectrophoresis confirmed the cross-reactivity of different psittacine sera with the anti-cockatiel IgG serum but not with ostrich and chicken sera. This antiserum detected antibody responses in sera from cockatiels vaccinated against chlamydial major outer membrane protein in an immunoblot assay.

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.

Thalidomide delayed the ability of 4T1 cells to amass into tumors in Balb/c mice.

Thalidomide (Thal) can suppress the growth of established, as well as explanted tumors in mice. We wanted to determine if it could suppress the ability of tumor cells to assemble and establish a primary tumor at the injection site. Using the mouse 4T1 mammary tumor model, we fed Thal to mice for 4 days, then injected 105 4T1 cells into the interscapular region of Balb/c mice. After 20 days on treatment with Thal, all seven control mice, fed with meal had tumors ranging from 3 to 93 mm3 (median 20). Two of the eight mice fed with meal + Thal had no tumors, and the remaining mice had tumors ranging from 2 to 22 mm3 (median 5). The median volume of the tumors in the control group was significantly more than that of mice treated with Thal (p = 0.03, Mann–Whitney test). In vitro treatment of the 4T1cells with Thal did not inhibit their ability to proliferate, to adhere to plastic, or to bind to Concanavalin-A. Thal caused a marked reduction in the ability of the 4T1 cells to assemble into palpable tumors.