Gary J. Nabel

The rapid spread of HIV as an emerging virus serves as a reminder of the continued susceptibility of large segments of the world population to infectious agents. HIV has adopted several different strategies by which to evade immune detection that have promoted its spread (Bagarazzi et al., 1998; Burton and Moore, 1998; Heilman and Baltimore, 1998). First, by attacking the CD4⁺ T cell, a major host cell that defends against foreign pathogens, the virus eliminates the specific cell type perhaps best suited for immune protection. Second, the virus encodes gene products that alter the immune recognition and activation. These viral proteins include Nef, which can decrease the expression of both CD4 and class I MHC molecules and participate in cell-cell interactions that lead to the detection of foreign antigen. Third, the virus undergoes rapid genetic mutation and has been selected to tolerate a number of changes at the nucleotide level and is therefore a “moving target” for immune recognition. Finally, persistent viral infection in HIV-seropositive individuals increases the duration of infection and the likelihood of transmission, particularly during its asymptomatic phase.

For these reasons, HIV has spread throughout the world, causing infection in more than 30 million people (Palella, 1998). Current projections estimate that an additional 5.8 million individuals are likely to become infected within the next year (UNAIDS, 1998). In addition, the virus has developed heterogeneity in different parts of the world, and it appears that specific forms predominate in certain populations. For exam-

ple, clade B is most common in the United States, whereas clades C and E are becoming prevalent in East Asia and Africa (Gao et al., 1996a). The development of new clades and continual genetic variability provide the basis for further genetic drift, migration, and infection of new populations. In some cases the characteristics of this virus alter its mode of spread; for example, there is some evidence for differences in sexual transmission patterns from clade to clade (Gao et al., 1996b). In addition to clade differences among HIV strains, two different HIV strains—HIV-1 and HIV-2—provide for further genetic diversity and potential recombination between related regions of these viruses. Thus, it is critical that appropriate detection and surveillance are maintained in order to understand the patterns of spread of different viral types. Effective surveillance will require appropriate cost-effective, high-throughput technology, which has not been fully developed. This information may be used to predict the future populations at risk and the potential for altered pathogenicity in these populations.

HIV provides an example relevant to the emergence of other viruses in the world population. Ebola virus represents another recently emerged virus responsible for several outbreaks in Africa, Europe, and North America (see Figure 8.1). These outbreaks have often been associated with infected primates, followed by person-to-person spread. Of great concern regarding Ebola virus infection is its high mortality rate, in some cases up to 90 percent. Several aspects of its biology raise these concerns. Although the virus is readily contained using barrier precautions, the reservoir for infection is currently unknown. The virus is poorly recognized by the immune system and is said to be nonimmunogenic, with no vaccines or treatments for it currently available. New technologies may soon make it possible to rapidly implement vaccination approaches for diseases such as Ebola and HIV. While HIV has proven to be a difficult target for vaccine development, preliminary studies with Ebola virus appear to be more encouraging. Improved technology, however, could help to contain the spread of both of these viruses.

In the case of Ebola virus, at the National Institutes of Health (NIH) we have used newer techniques involving DNA immunization to control infection and have found that the use of plasmid expression vectors that encode Ebola virus gene products can be used for successful genetic immunization in rodent models. In a guinea pig model that resembles human disease, immunization induces both humoral immunity and cellular immunity that confer protection against lethal challenge by the

virus (Xu et al., 1998). It appears that T-cell-immunity is required for this protection, as measured by an indirect immune parameter, the antibody response to the viral glycoprotein (GP), a T-cell dependent response. Animals that have titers greater than 1:5,000 are nearly completely protected when exposed to a lethal dose of the virus that otherwise causes mortality within a week in unprotected recipients (see Table 8.1). Recently, this approach has been applied, with an adenoviral vector boost, to confer protective immunity against lethal Ebola virus challenge in nonhuman primates (Sullivan et al., 2000).

These approaches are being applied by many laboratories to HIV and to a number of other infectious agents, including tuberculosis, influenza virus, malaria, and herpesvirus (Tighe et al., 1998; Donnelly et al., 1997). Despite promise for many pathogens, difficulties remain in generating successful vaccines for HIV. Such limitations point to a number of stumbling blocks for vaccine development, an area in which bioinformatics and genetic analyses may provide new opportunities and prove useful.

An illustration of the complexity and unpredictability of immune responses to foreign proteins comes from our studies of the immune response to different viral glycoproteins (see Figure 8.2). In the case of Ebola virus as well as HIV, specific viral genes induce characteristic immune responses that are specific to particular proteins. For example, DNA immunization with expression vectors encoding gp160 readily induces a cytolytic T-cell response to this protein but fails to induce a robust antibody response (data not shown). In contrast, vaccination to another HIV gene product, Nef, which plays an important role in increasing the pathogenicity of infection and the onset of AIDS symptoms, leads to the generation of excellent humoral immunity but poor cytolytic T-cell responses. Analogous observations have been made in other systems. For example, the Ebola virus glycoprotein readily induces cytolytic T-cell responses but does not induce high-titer antibody responses. In contrast, the nucleoprotein can induce titers greater than 1:60,000 in mice but stimulates

FIGURE 8.2 Inherent differences in intrinsic immune responses to Ebola gene products.

essentially no cytolytic T-cell response (Xu et al., 1998). The nature of these immune responses is not random. In fact, it is likely that these viruses have evolved such gene products not only to facilitate viral replication but also to evade immune detection. In Ebola virus infection, the virus displays high replication potential and is likely to rely on innate immune and inflammatory responses to limit spread (Baize et al., 1999; Nabel,

1999), whereas lentiviruses cause a more indolent disease course, with immune mechanisms playing a more important role.

The challenge is to use information technologies to understand the mechanisms by which specific viral genes induce their characteristic immune responses by using different vaccine strategies and to develop algorithms by which these immune responses can be predicted. The development of approaches by which specific peptides can be made more immunogenic in a predictable fashion using alternative vectors or adjuvants in humans will facilitate the development of effective vaccines and lead to more rational and efficient vaccine approaches. HIV gp160 and Nef provide an opportunity to undertake such analyses. The primary amino acid sequences of these proteins are known, and deletion-mapping studies can be performed, making it possible to define motifs that may be responsible for generating specific immune reactivity. In addition, known motifs promote targeting of proteins to specific cell compartments. These include myristoylation sites, signal sequences, and endoplasmic reticulum trafficking signals (glycosylation motifs). Through a combination of modeling, motif recognition, and the application of bioinformatics and empirical testing, it should be possible to better define the underlying principles that govern these responses. Ideally, such efforts may allow prediction of immune responses to specific amino acid sequences and improved efficacy in the design of effective immunogens for vaccine development. Such vaccines could be rapidly synthesized by recombinant DNA techniques and formulated to respond in a timely fashion to infectious outbreaks. Although this technology will not provide the first line of defense in containing viral infection, it may provide an opportunity to immunize populations more distant from sites of initial exposure and thus contain disease.

Finally, these recombinant techniques also provide the opportunity for more rapid development of antiviral treatment for human infectious disease. In the case of Ebola virus we have learned that expression of the viral glycoprotein can lead to severe cytopathic effects in cell culture models in this disease (Yang et al., 2000). Synthesis of the Ebola virus glycoprotein causes severe cellular cytotoxicity when expressed in endothelial cells in the laboratory. This glycoprotein mediates this effect

through a specific domain of the protein. When this domain is deleted, its toxicity is eliminated. We have been able to map the genetic determinants that lead to cellular cytotoxicity rapidly, and it should be possible to use this information to identify the mechanism of such viral cytopathic effects. During infectious outbreaks where disease mortality is high, it may be possible to limit the mortality of the disease rather than eliminate the infection entirely to prevent its lethality in large populations. In the case of Ebola virus we have begun to identify drugs that interfere with the synthesis of this protein. Additional studies will provide further insight into its molecular mechanism. These mechanisms of cytotoxicity that apply to Ebola virus may well apply to other hemorrhagic fevers and could provide for generic classes of drugs that may be useful in blunting the lethality of an ongoing outbreak.

Molecular genetics, information technologies, and bioengineering can significantly improve efforts to prevent, detect, and treat infectious diseases. It is likely that improved technologies for data collection, molecular analysis, and recombinant genetics will facilitate our ability to contain outbreaks of both naturally occurring and iatrogenic infections. Implementation of cost-effective, rapid, high-throughput screening technology at remote sites with information transfer to diverse locations for data analysis by appropriate epidemiologic and scientific personnel can allow for more expedient and effective responses to emerging infectious threats.

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Yang, Z., H. J. Duckers, N. J. Sullivan, A. Sanchez, E. G. Nabel, and G. J. Nabel. 1999. Identification of the Ebola virus glycoprotein as the main viral determinant of vascular cell cytotoxicity and injury. Nature Medicine, 6:886-889.