Quantification of regional aerosol deposition patterns as a function of aerodynamic particle size in rhesus macaques using PET/CT imaging.

Aerosol aerodynamic particle size is known to affect deposition patterns of inhaled aerosol particles, as well as the virulence of inhaled bioaerosol particles. While a significant amount of work has been performed to describe the deposition of aerosol particles in the human respiratory tract, only a limited amount of work has been performed to describe the deposition of aerosol particles in the respiratory tract of nonhuman primates, an animal model commonly utilized in pharmacological and toxicological studies, especially in the biodefense field. In this study, anesthetized rhesus macaques inhaled radiolabeled aerosols with MMADs of 1.7, 3.6, 7.4 and 11.8 µm to characterize regional deposition patterns. The results demonstrate that the regional deposition pattern shifts as particle size increases, with greater deposition in more proximal regions of the respiratory tract and decreased deposition in the pulmonary region. The results of this study extend the findings of previous studies which demonstrated a similar shift in the deposition pattern as a function of particle size by providing greater resolution of deposition patterns. These data on regional deposition patterns provide a starting point to begin to explore potential mechanisms responsible for the differences in virulence of infectious bioaerosols as a function of particle size and deposition pattern reported in previous studies. Additionally, the data are useful to assess the performance of various deposition models that have been published in the literature.

Marburg virus vaccines: comparing classical and new approaches.

An effort to develop a safe and effective vaccine for Marburg virus (MBGV), one of the filoviruses known to cause high mortality rates in humans, led us to compare directly some of the merits of modern versus classical vaccine approaches for this agent. Prior work had established the MBGV-glycoprotein (GP), the only known virion surface antigen, as a candidate for inclusion in a vaccine. In this study, we vaccinated groups of Hartley guinea pigs with killed MBGV, live attenuated MBGV, soluble MBGV-GP expressed by baculovirus recombinants, MBGV-GP delivered as a DNA vaccine, or MBGV-GP delivered via an alphavirus RNA replicon. Serological responses were evaluated, and animals were challenged with a lethal dose of MBGV given either subcutaneously or via aerosol. Killed MBGV and replicon-delivered MBGV-GP were notably immunogenic and protective against MBGV, but results did not exclude any approach and suggested a role for DNA vaccines in immunological priming.

Epitopes involved in antibody-mediated protection from Ebola virus.

To determine the ability of antibodies to provide protection from Ebola viruses, monoclonal antibodies (mAbs) to the Ebola glycoprotein were generated and evaluated for efficacy. We identified several protective mAbs directed toward five unique epitopes on Ebola glycoprotein. One of the epitopes is conserved among all Ebola viruses that are known to be pathogenic for humans. Some protective mAbs were also effective therapeutically when administered to mice 2 days after exposure to lethal Ebola virus. The identification of protective mAbs has important implications for developing vaccines and therapies for Ebola virus.

Marburg virus vaccines based upon alphavirus replicons protect guinea pigs and nonhuman primates.

Marburg virus (MBGV), for which no vaccines or treatments currently exist, causes an acute hemorrhagic fever with a high mortality rate in humans. We previously showed that immunization with either killed MBGV or a glycoprotein (GP) subunit prevented lethal infection in guinea pigs. In the studies reported here, an RNA replicon, based upon Venezuelan equine encephalitis (VEE) virus, was used as a vaccine vector; the VEE structural genes were replaced by genes for MBGV GP, nucleoprotein (NP), VP40, VP35, VP30, or VP24. Guinea pigs were vaccinated with recombinant VEE replicons (packaged into VEE-like particles), inoculated with MBGV, and evaluated for viremia and survival. Results indicated that either GP or NP were protective antigens while VP35 afforded incomplete protection. As a more definitive test of vaccine efficacy, nonhuman primates (cynomolgus macaques) were inoculated with VEE replicons expressing MBGV GP and/or NP. Three monkeys received packaged control replicons (influenza HA); these died 9 or 10 days after challenge, with typical MBGV disease. MBGV NP afforded incomplete protection, sufficient to prevent death but not disease in two of three macaques. Three monkeys vaccinated with replicons which expressed MBGV GP, and three others vaccinated with both replicons that expressed GP or NP, remained aviremic and were completely protected from disease.

Quantitative studies of heteropolymer-mediated binding of inactivated Marburg virus to the complement receptor on primate erythrocytes.

Previous in vitro and in vivo experiments in our laboratory have demonstrated that cross-linked bispecific monoclonal antibody (mAb) complexes (Heteropolymers, HP) facilitate binding of prototype pathogens to primate erythrocytes (E) via the E complement receptor, CR1. These E-bound immune complexes are safely and rapidly cleared from the bloodstream. In order to generate a robust bispecific system for HP-mediated clearance of real pathogens such as Filoviruses, we have developed the necessary methodologies and reagents using both inactivated Marburg virus (iMV) and a recombinant form of its surface envelope glycoprotein (rGP). We identified mAbs which bind rGP in solution phase immunoprecipitation experiments. HP were prepared by chemically cross-linking an anti-CR1 mAb with several of these anti-Marburg virus mAbs and used to facilitate binding of iMV and rGP to monkey and human E. These HP mediate specific and quantitative binding (> or = 90%) of both antigens to monkey and human E. Binding was also demonstrable in an indirect RIA. E with bound Marburg virus were probed with 125I labeled mAbs to the Marburg surface glycoprotein and more than 100 mAbs are bound per E. It should be possible to adapt this general approach to other pathogens, and experiments underway should lead to an in vivo test of HP-mediated clearance of Marburg virus.

Antigenicity and vaccine potential of Marburg virus glycoprotein expressed by baculovirus recombinants.

There is no effective vaccine for Marburg virus (MBGV) or any other filovirus, nor enough pertinent information to expedite rational vaccine development. To ascertain some of the minimal requirements for a MBGV vaccine, we determined whether whole inactivated MBGV, or a baculovirus-expressed virion subunit, could be used to immunize guinea pigs against a lethal infection. Baculovirus recombinants were made to express the MBGV glycoprotein (GP) either as a full-length, cell-associated molecule or a slightly truncated (5.4%) product secreted into medium; the latter, for its far greater ease in manipulation, was tested for its vaccine potential. Like MBGV GP, both the full-length and truncated GP expressed by baculovirus recombinants were abundantly glycosylated with both N- and O-linked glycans; differences in glycosylation were detectable, but these could not be shown to affect antigenicity with respect to available antibodies. The recombinant truncated glycoprotein elicited protection against lethal challenge with the MBGV isolate from which it was constructed and less effectively against an antigenically disparate MBGV isolate. Killed (irradiated) MBGV antigen was protective, in a reciprocal fashion, against both MBGV types. In a preliminary assessment of possible protective mechanisms, serum antibodies from immune animals were shown to be sufficient for protecting naive guinea pigs from lethal MBGV infections

Complementation of human immunodeficiency virus type 1 vif mutants in some CD4+ T-cell lines.

The viral infectivity factor gene, vif of human immunodeficiency virus type 1 (HIV-1), is required for full infectivity in most T-cell lines. The replication kinetics exhibited by these mutants has been shown to be cell type-dependent. In H9 cells as well as primary lymphocytes, vif mutants are incapable of establishing infection. This has led to classification of these cell types as non-permissive for vif mutant replication. The T-cell lines Sup T1 and C8166 are able to replicate the vif mutant virus, leading to their classification as permissive for vif mutant replication. In this study, four cell lines (Sup T1, C8166, Molt 4 Clone 8, and A3.01) were tested for their ability to replicate vif mutant virus derived from two different strains of HIV-1 (HXB2 and NL4-3) that had been passaged on various cell lines. Although the kinetics of initial infection was delayed in all cells, by the second passage of vif mutant virus on Sup T1 or Molt 4 cells the kinetics of replication were identical to wild type virus. In contrast, mutant virus displayed delayed replication kinetics in C8166 and A3.01 cells in both initial and subsequent passages. In addition, the levels of viral DNA in infected Sup T1 cells were similar for delta vif and wild type virus, but in C8166 cells delta vif virus DNA levels were reduced compared to wild type virus. These results argue that in Sup T1 and Molt 4 cells there is a factor present that is able to complement the defect in vif mutant viruses which is absent or inefficient in its activity in C8166 and A3.01 cells.