William H. Wunner

Management of Rabies in Humans

Rabies is a fatal disease in humans, and, to date, the only survivors of the disease have received rabies vaccine before the onset of illness. The approach to management of the rabies normally should be palliative. In unusual circumstances, a decision may be made to use an aggressive approach to therapy for patients who present at an early stage of clinical disease. No single therapeutic agent is likely to be effective, but a combination of specific therapies could be considered, including rabies vaccine, rabies immunoglobulin, monoclonal antibodies, ribavirin, interferon-alpha, and ketamine. Corticosteroids should not be used. As research advances, new agents may become available in the future for the treatment of human rabies.

Influenza A virus infection engenders a poor antibody response against the ectodomain of matrix protein 2

BackgroundMatrix protein 2 (M2) is an integral tetrameric membrane protein of influenza A virus (IAV). Its ectodomain (M2e) shows remarkably little diversity amongst human IAV strains. As M2e-specific antibodies (Abs) have been shown to reduce the severity of infection in animals, M2e is being studied for its capability of providing protection against a broad range of IAV strains. Presently, there is little information about the concentration of M2e-specific Abs in humans. Two previous studies made use of ELISA and Western blot against M2e peptides and recombinant M2 protein as immunosorbents, respectively, and reported Ab titers to be low or undetectable. An important caveat is that these assays may not have detected all Abs capable of binding to native tetrameric M2e. Therefore, we developed an assay likely to detect all M2e tetramer-specific Abs.ResultsWe generated a HeLa cell line that expressed full length tetrameric M2 (HeLa-M2) or empty vector (HeLa-C10) under the control of the tetracycline response element. These cell lines were then used in parallel as immunosorbents in ELISA. The assay was standardized and M2e-specific Ab titers quantified by means of purified murine or chimeric (mouse variable regions, human constant regions) M2e-specific Abs in the analysis of mouse and human sera, respectively. We found that the cell-based ELISA was substantially more effective than immobilized M2e peptide in detecting M2e-specific Abs in sera of mice that had recovered from repetitive IAV infections. Still, titers remained low (< 5 μg/ml) even after two consecutive infections but increased to ~50 μg/ml after the third infection. Competition with free M2e peptide indicated that ~20% of M2e-specific Abs engendered by infection reacted with M2e peptide. In humans presenting with naturally acquired influenza virus infection, 11 of 24 paired sera showed a ≥ 4-fold increase in M2e-specific Ab titer. The Ab response appeared to be of short duration as titers were very low (average 0.2 μg/ml) in all patients at onset of infection and in controls, in spite of evidence for previous exposure to IAV.ConclusionThe results provide convincing evidence that M2e-specific Ab-mediated protection is currently lacking or suboptimal in humans.

Bin1 functionally interacts with Myc and inhibits cell proliferation via multiple mechanisms

The tumor suppressor Bin1 was identified through its interaction with the N-terminal region of Myc which harbors its transcriptional activation domain. Here we show that Bin1 and Myc physically and functionally associate in cells and that Bin1 inhibits cell proliferation through both Myc-dependent and Myc-independent mechanisms. Bin1 specifically inhibited transactivation by Myc as assayed from artificial promoters or from the Myc target genes ornithine decarboxylase (ODC) and α prothymosin (pT). Inhibition of ODC but not pT required the presence of the Myc binding domain (MBD) of Bin1 suggesting two mechanisms of action. Consistent with this possibility, a non-MBD region of Bin1 was sufficient to recruit a repression function to DNA that was unrelated to histone deacetylase. Regions outside the MBD required for growth inhibition were mapped in Ras cotransformation or HepG2 hepatoma cell growth assays. Bin1 required the N-terminal BAR domain to suppress focus formation by Myc whereas the C-terminal U1 and SH3 domains were required to inhibit adenovirus E1A or mutant p53, respectively. All three domains contributed to Bin1 suppression of tumor cell growth but BAR-C was most crucial. These findings supported functional interaction between Myc and Bin1 in cells and indicated that Bin1 could inhibit malignant cell growth through multiple mechanisms.

Localization and immunological characterization of antigenic domains of the rabies virus internal N and NS proteins

To locate epitopes on internal antigens of rabies virus, purified N and NS proteins of the nucleocapsid were cleaved at methionine, tryptophan or glutamic acid residues, transferred to nitrocellulose and immunostained using monoclonal antibodies (MAbs) specific for N and NS proteins, respectively. Five MAb-positive fragments of N protein and one fragment of NS protein were located after NH2-terminal amino acid sequence analysis within the deduced amino acid sequences of N and NS proteins. Antigenic analysis of synthetic overlapping peptides corresponding to the amino acid sequences of these fragments localized two major antigenic sites of N protein and one antigenic site of NS protein. Like the N- and NS-specific MAbs, anti-peptide antisera produced against the different synthetic antigens either reacted in a type-common fashion with all rabies virus strains, or in a type-specific manner with a restricted number of strains. The synthetic peptides corresponding to the three antigenic regions of the N and NS proteins also stimulated proliferation of human T lymphocytes derived from vaccinees who received inactivated rabies virus vaccine. This suggested that the antigenic regions of N and NS proteins are recognized by both B and T cells.

Chemical and immunological analysis of the rabies soluble glycoprotein.

Soluble glycoprotein (Gs), purified from virion-depleted, rabies-infected tissue culture fluid, was chemically and immunologically analyzed. A comparison of this antigen with the virion-associated glycoprotein showed that Gs lacks 58 amino acid residues from the carboxy terminus of the virion-associated glycoprotein. Analysis with monoclonal antibodies revealed that all the epitopes of the viral glycoprotein are also present in the soluble glycoprotein. However, when tested for its ability to protect mice against a lethal challenge infection with rabies virus, Gs in contrast to viral glycoprotein, showed no protective activity. These results suggest that the carboxy terminus of the rabies virus glycoprotein is necessary for its full protective activity even though this portion of the glycoprotein molecule does not contain any antigenic determinants.

RABIES SUBUNIT VACCINES

Conclusions The secondary and tertiary structures of the rabies virus spike G protein are important for its ability to induce VN antibodies and confer immunity to the host. For a subunit peptide vaccine to be as effective as the native spike G protein, it would appear that the amino acid sequence comprising the antigenic determinant for VN antibody binding must be made to fold properly even when deprived of its native support structure. Since CNBr peptides have retained at least some of their antigenicity for binding antibodies from hyperimmune serum but not monoclonal VN antibodies, and their immunogenicity, then synthetic peptides containing corresponding sequences should show similar activities. Additionally, determinants that might be necessary for stimulating T lymphocytes would have to be built into the synthetic peptide preparation. It would also appear that a properly folded peptide might have to be aggregated into suitably large particles for it to achieve its full protective effect. Adjuvants may serve in this capacity to enhance the immune response to relevant peptides and thus improve the immunogenicity of a subunit vaccine that ultimately protects animals and humans against rabies virus infection.

Mechanisms of rabies virus neutralization by glycoprotein-specific monoclonal antibodies.

Incubation of radiolabeled rabies virus with neutralizing monoclonal antibodies (MAbs) resulted in complete neutralization of the virus but only partial inhibition of virus binding to, and internalization by, BHK cells. Several of the neutralizing MAbs were capable of preventing infection after virus adsorption to cells; up to 30% of the bound virus was released when cells containing adsorbed virus were incubated with these MAbs at 4 degrees, indicating that the release of bound virus accounts only in part for the neutralization of adsorbed virus. To study the mechanism of neutralization of cell-bound virus, temperature shift experiments were carried out to follow the fate of neutralized cell-adsorbed virus at 37 degrees. Treatment of infected cells with each of the tested neutralizing MAbs had no effect on virus uptake at 37 degrees and the MAbs were endocytosed together with the virus; however, the ability of some of the MAbs to neutralize cell-adsorbed rabies virus correlated with the fusion inhibition activity of these MAbs. We hypothesize from these data that these MAbs neutralize rabies virus by inhibiting the intraendosomal acid-catalyzed fusion step that leads to virus uncoating.

Rabies in the 21st Century

Why are 50,000–55,000 people dying from rabies worldwide each year, with 25,000–30,000 human deaths in India alone and over 3 billion people continuing to be at risk of rabies virus infection in over 100 countries in the 21st century? These are astonishing numbers, particularly as they represent individuals, a large proportion of whom are children, who have been attacked or are likely to be attacked by rabid dogs, the main source of rabies virus infection that, as yet, has not been brought under control in many parts of the world. The number of human deaths and the circumstances by which these deaths continue to occur are extraordinary, with over 95% of rabies victims reported residing in Asia and Africa and nearly all victims of a rabid dog bite. Rabies has been part of the history of civilization for several millennia, rooted in its enzootic environment (animal host) and causing severe threats to public health across continents. Rabies and the symptoms it presents can hardly be ignored, yet it appears to be unduly neglected in some parts of the world, notably in Asia and Africa, where the spread of canine rabies is not under control and is far from being eliminated. In other parts of the world, largely in developed countries, where elimination of canine rabies has been achieved, there are models to be followed and lessons learned that will challenge epidemiologists and molecular virologists alike in the future as they apply new techniques to achieve the elimination of canine and human rabies worldwide.

Characterization of a new virus-neutralizing epitope that denotes a sequential determinant on the rabies virus glycoprotein.

Two new monoclonal antibodies (MAbs) derived from mice immunized with the Pitman-Moore (PM) strain of rabies virus were used to identify and characterize two unique antigenic determinants on the rabies virus glycoprotein. One of the determinants, which defined an additional antigenic site on the rabies virus glycoprotein, was delineated as a distinct epitope by the newly generated MAb, 6-15C4, in competitive binding studies and by comparative antigenic analysis of neutralization-resistant variant viruses. Both antigenic determinants were compared with the five previously described antigenic sites which bind virus-neutralizing antibodies on the challenge virus standard (CVS) and Evelyn-Rokitnicki-Abelseth (ERA) strain glycoproteins. The results presented in this communication show that the 6-15C4 epitope is the first epitope described in the rabies virus glycoprotein that does not depend on the native conformation of the glycoprotein for binding virus-neutralizing antibody. These data suggest that it may be possible to generate a synthetic peptide vaccine against rabies.

Oral vaccination of racoons (Procyon lotor) with baculovirus-expressed rabies virus glycoprotein

Successful field oral vaccination and protection against viral diseases have so far been achieved only with live-attenuated or live-recombinant virus vaccines. In this communication, we present data that demonstrate that a glycoprotein derived from recombinant baculovirus-infected insect cells is efficacious as an oral vaccine. The glycoprotein (G) of rabies virus (Evelyn Rokitnicki Abelseth strain) was abundantly expressed in a baculovirus expression system and oral vaccination of racoons with the baculovirus-expressed G protein resulted in the production of rabies virus-neutralizing antibodies and protection against a lethal challenge with a street rabies virus. The potential for using the baculovirus-expressed G protein for oral immunization of wildlife is discussed.