21 research outputs found
Production and Characterization of Human Monoclonal Antibody Fab Fragments to Vaccinia Virus from a Phage-Display Combinatorial Library
No full textA combinatorial, phage-display library of human Fab antibody fragments was generated from IgG heavy chain (HC) and light chain (LC) genes cloned from the lymphocytes of a vaccinia virus (VACV)-immune donor. To ascertain the complexity of the library, nucleotide sequences of the variable regions of the HC and LC genes were determined. Fourteen distinct HC and 18 distinct LC (7 κ and 11 λ) that formed a combinatorial library of 22 Fabs were identified. Immune-precipitation of radiolabeled VACV revealed that at least six different VACV proteins were recognized by the antibodies. Plaque-reduction neutralization demonstrated that six of the Fabs neutralized VACV in the presence of anti-human antibody. ELISA studies indicated that 15 of the Fabs were cross-reactive with monkeypox virus
Production and Characterization of Human Monoclonal Antibody Fab Fragments to Vaccinia Virus from a Phage-Display Combinatorial Library
No full textAbstractA combinatorial, phage-display library of human Fab antibody fragments was generated from IgG heavy chain (HC) and light chain (LC) genes cloned from the lymphocytes of a vaccinia virus (VACV)-immune donor. To ascertain the complexity of the library, nucleotide sequences of the variable regions of the HC and LC genes were determined. Fourteen distinct HC and 18 distinct LC (7 κ and 11 λ) that formed a combinatorial library of 22 Fabs were identified. Immune-precipitation of radiolabeled VACV revealed that at least six different VACV proteins were recognized by the antibodies. Plaque-reduction neutralization demonstrated that six of the Fabs neutralized VACV in the presence of anti-human antibody. ELISA studies indicated that 15 of the Fabs were cross-reactive with monkeypox virus
Pathogenic Hantaviruses Elicit Different Immunoreactions in THP-1 Cells and Primary Monocytes and Induce Differentiation of Human Monocytes to Dendritic-Like Cells
No full textHantaviruses cause two important human illnesses, hemorrhagic fever with renal syndrome (HFRS) and hantavirus
pulmonary syndrome (HPS). Both syndromes are believed to be immune-mediated diseases. Monocytes/macrophages are
thought to be the main target cells for hantaviruses and important sources of and targets for cytokines/chemokines secretion.
THP-1 cells have been used extensively as models for primary monocytes in biocompatibility research. The aim of
our study was to determine if hantaviruses induce the same immunoreactions in THP-1 cells and primary monocytes/
macrophages and might therefore be suitable for immune studies of hantaviral infections. For that purpose we compared
various cytokines/chemokines and their receptors in THP-1 cell line and primary monocytes/macrophages. Infected primary
monocytes/macrophages induced mostly -chemokines and their receptors. In contrast, THP-1 cells, expressed receptors
for CXC chemokines. Surprisingly, infected macrophages underwent morphological changes toward dendriticlike
cells and increased expression of co-stimulatory molecules: CD40, CD80, CD83 and CD86. Our data indicate that
THP-1 cells are not ideal for in vitro research of the immunopathogenesis of hantaviruses in humans. Further, our studies
revealed potential roles for cytokines/chemokines in HFRS/HPS immunopathogenesis and point to intriguing possibilities
for the possible differentiation of infected macrophages to dendritic-like cells
Immunogenicity of combination DNA vaccines for Rift Valley fever virus, tick-borne encephalitis virus, Hantaan virus, and Crimean Congo hemorrhagic fever virus
No full textDNAvaccines for RiftValley fever virus (RVFV), Crimean Congo hemorrhagic fever virus (CCHFV), tick-borne encephalitis virus (TBEV), and Hantaan virus (HTNV), were tested in mice alone or in various combinations. The bunyavirus vaccines (RVFV, CCHFV, and HTNV) expressed Gn and Gc genes, and the flavivirus vaccine (TBEV) expressed the preM and E genes. All vaccines were delivered by gene gun. The TBEV DNA vaccine and the RVFV DNA vaccine elicited similar levels of antibodies and protected mice from challenge when delivered alone or in combination with other DNAs. Although in general, the HTNV and CCHFV DNA vaccines were not very immunogenic in mice, there were no major differences in performance when given alone or in combination with the other vaccine
Mixing of M segment DNA vaccines to Hantaan virus and Puumala virus reduces their immunogenicity in hamsters
No full textTo determine if DNA vaccines for two hantaviruses causing hemorrhagic fever with renal syndrome, Hantaan virus and Puumala virus, are immunogenic when given in combination, we delivered them to hamsters separately or as mixtures by gene gun or by electroporation. Both vaccines elicited neutralizing antibodies when given alone but when theywere delivered as a mixture, antibodies to only one of the two hantaviruses could be detected. In contrast, if the DNAs were given as separate vaccinations to a single animal, responses to both were observed. These studies suggest that the two DNA vaccines will need to be given as separate administrations
PLoS Negl Trop Dis
No full textFor enveloped viruses, fusion of the viral envelope with a cellular membrane is critical for a productive infection to occur. This fusion process is mediated by at least three classes of fusion proteins (Class I, II, and III) based on the protein sequence and structure. For Rift Valley fever virus (RVFV), the glycoprotein Gc (Class II fusion protein) mediates this fusion event following entry into the endocytic pathway, allowing the viral genome access to the cell cytoplasm. Here, we show that peptides analogous to the RVFV Gc stem region inhibited RVFV infectivity in cell culture by inhibiting the fusion process. Further, we show that infectivity can be inhibited for diverse, unrelated RNA viruses that have Class I (Ebola virus), Class II (Andes virus), or Class III (vesicular stomatitis virus) fusion proteins using this single peptide. Our findings are consistent with an inhibition mechanism similar to that proposed for stem peptide fusion inhibitors of dengue virus in which the RVFV inhibitory peptide first binds to both the virion and cell membranes, allowing it to traffic with the virus into the endocytic pathway. Upon acidification and rearrangement of Gc, the peptide is then able to specifically bind to Gc and prevent fusion of the viral and endocytic membranes, thus inhibiting viral infection. These results could provide novel insights into conserved features among the three classes of viral fusion proteins and offer direction for the future development of broadly active fusion inhibitors
Evaluation of Tick-Borne Encephalitis DNA Vaccines in Monkeys
No full textAbstractTick-borne encephalitis is usually caused by infection with one of two flaviviruses: Russian spring summer encephalitis virus (RSSEV) or Central European encephalitis virus (CEEV). We previously demonstrated that gene gun inoculation of mice with naked DNA vaccines expressing the prM and E genes of these viruses resulted in long-lived homologous and heterologous protective immunity (Schmaljohn et al., 1997). To further evaluate these vaccines, we inoculated rhesus macaques by gene gun with the RSSEV or CEEV vaccines or with both DNA vaccines and compared resulting antibody titers with those obtained by vaccination with a commercial, formalin-inactivated vaccine administered at the human dose. Vaccinations were given at days 0, 30, and 70. All of the vaccines elicited antibodies detected by ELISA and by plaque-reduction neutralization tests. The neutralizing antibody responses persisted for at least 15 weeks after the final vaccination. Because monkeys are not uniformly susceptible to tick-borne encephalitis, the protective properties of the vaccines were assessed by passive transfer of monkey sera to mice and subsequent challenge of the mice with RSSEV or CEEV. One hour after transfer, mice that received 50 μl of sera from monkeys vaccinated with both DNA vaccines had circulating neutralizing antibody levels <20–80. All of these mice were protected from challenge with RSSEV or CEEV. Mice that received 10 μl of sera from monkeys vaccinated with the individual DNA vaccines, both DNA vaccines, or a commercial vaccine were partially to completely protected from RSSEV or CEEV challenge. These data suggest that DNA vaccines may offer protective immunity to primates similar to that obtained with a commercial inactivated-virus vaccine
DNA Vaccines Expressing either the GP or NP Genes of Ebola Virus Protect Mice from Lethal Challenge
No full textAbstractDNA vaccines expressing the envelope glycoprotein (GP) or nucleocapsid protein (NP) genes of Ebola virus were evaluated in adult, immunocompetent mice. The vaccines were delivered into the skin by particle bombardment of DNA-coated gold beads with thePowderJect-XRgene gun. Both vaccines elicited antibody responses as measured by ELISA and elicited cytotoxic T cell responses as measured by chromium release assays. From one to four vaccinations with 0.5 μg of the GP DNA vaccine resulted in a dose-dependent protection from Ebola virus challenge. Maximal protection (78% survival) was achieved after four vaccinations. Mice were completely protected with a priming dose of 0.5 μg of GP DNA followed by three or four subsequent vaccinations with 1.5 μg of DNA. Partial protection could be observed for at least 9 months after three immunizations with 0.5 μg of the GP DNA vaccine. Comparing the GP and NP vaccines indicated that approximately the same level of protection could be achieved with either vaccine
The genetic adjuvant IL-12 enhances the protective efficacy of a DNA vaccine for Venezuelan equine encephalitis virus delivered by intramuscular injection in mice
No full textEnhanced Efficacy of a Codon-Optimized DNA Vaccine Encoding the Glycoprotein Precursor Gene of Lassa Virus in a Guinea Pig Disease Model When Delivered by Dermal Electroporation
No full textLassa virus (LASV) causes a severe, often fatal, hemorrhagic fever endemic to West Africa. Presently, there are no FDA-licensed medical countermeasures for this disease. In a pilot study, we constructed a DNA vaccine (pLASV-GPC) that expressed the LASV glycoprotein precursor gene (GPC). This plasmid was used to vaccinate guinea pigs (GPs) using intramuscular electroporation as the delivery platform. Vaccinated GPs were protected from lethal infection (5/6) with LASV compared to the controls. However, vaccinated GPs experienced transient viremia after challenge, although lower than the mock-vaccinated controls. In a follow-on study, we developed a new device that allowed for both the vaccine and electroporation pulse to be delivered to the dermis. We also codon-optimized the GPC sequence of the vaccine to enhance expression in GPs. Together, these innovations resulted in enhanced efficacy of the vaccine. Unlike the pilot study where neutralizing titers were not detected until after virus challenge, modest neutralizing titers were detected in guinea pigs before challenge, with escalating titers detected after challenge. The vaccinated GPs were never ill and were not viremic at any timepoint. The combination of the codon-optimized vaccine and dermal electroporation delivery is a worthy candidate for further development
