The invention relates to glycoprotein B segments of herpes simplex virus types 1 and 2 (HSV-1, HSV-2) containing linear antigenic epitopes reactive with human antibodies to HSV-1 and HSV-2. Of particular interest are HSV glycoprotein B segments containing type-specific epitopes useful in serodiagnostic immunoassays for distinguishing HSV-1 infection from HSV-2 in humans, and in human vaccines for generating neutralizing antibodies to HSV-1 or HSV-2.
1. Field of Art
The herpes simplex virus (HSV) glycoprotein B (gB) is a transmembrane envelope glycoprotein that contributes to the penetration of virions into host cells by host cell recognition, viral adherence, and virion-cell membrane fusion. gB induces fusion of the virion envelope with the cellular cytoplasmic membrane, an essential function for virus entry. Immunization of humans and animals with purified gB induces virus-neutralizing antibody responses, and recombinant gB proteins are being investigated as HSV vaccines. The amino acid sequences for both gB-1 and gB-2 and their encoding nucleotide sequences are known and available, e.g., from Genebank.
The HSV type 2 gB (gB-2) polypeptide contains 904 amino acids (aa)--(FIG. 5). The amino-terminal segment (1-22aa) is a signal peptide that is cleaved from the mature protein during processing. aa 745 to 798 constitute a hydrophobic transmembrane anchor domain. The segment carboxy-proximal to the anchor domain (aa 799 to 904) is cytoplasmic and appears to mediate membrane fusion. The amino-proximal segment (aa 23 to 744) is extracellular and contains epitopes that are recognized by virus-neutralizing antibodies. HSV type 1 gB (gB-1) is structurally similar (FIG. 4).
HSV-2 and HSV-1 are closely related viruses. Most of their proteins, including gB, are highly conserved (very homologous) and are known to elicit cross-reactive antibody responses. It has accordingly been difficult to provide reliable, sensitive immunoassays capable of distinguishing HSV-1 from HSV-2 infection, or to provide type-specific vaccines effective against HSV infection.
2. Discussion of Related Art
HSV infections in humans are commonly diagnosed by immunoassay of blood samples for HSV antibodies in Western blot assays or other immunoassays using random lysates of cells infected with HSV-1 or HSV-2 as antigen targets. These serodiagnostic assays have been recognized as generally unsatisfactory for distinguishing HSV-1 infection from HSV-2 infection owing to strong responses of type-common antibody-reactive regions of both native proteins. This has proved to be a particular problem in distinguishing acute HSV-2 infection in patients previously infected with HSV-1, because of a likely anamnestic response to HSV-1 and HSV-2 type-common antigens which obscures responses to type-specific antigens. Difficulties in detecting HSV-2 type-specific antibodies by using HSV-2 cell lysate Western blot assays on subjects previously infected with HSV-1, for example, may result in a not uncommon misclassification of samples subjected to these assays as HSV-1 positive and HSV-2 negative. Accordingly, on-going research has attempted to identify domains of various native HSV viral proteins which are highly reactive with anti HSV-1 and anti HSV-2 antibodies and which provide a virus type-specific antibody response.
While several HSV proteins have been considered as sources of useful antigenic epitopes for diagnosis and prevention, only HSV-1 and HSV-2 glycoprotein G (gG-1, gG-2) has been found useful to date for use in assays with improved sensitivities and specificities. Most HSV-2 and HSV-1 proteins are very homologous, as noted above. However, HSV-2 gG-2 and HSV-1 gG-1 have highly dissimilar amino acid sequences, and human gG-2 and gG-1 antibody responses in the presence of HSV-2 and HSV-1 infections appear to have significantly improved virus type-specificity over lysates of infected cells. Thus, detection of antibody reactivities to native or recombinant HSV-1 or HSV-2 glycoprotein G presently forms the basis for current HSV type-specific immunoassays.
HSV glycoprotein B has also sparked interest as a possible source of type-specific HSV antigens. To date, however, only the complete proteins have been used, and these have exhibited only cross-reactive antibody responses. Virus-specific determinates to human antibodies have been eagerly sought for many years, without notable success.
HSV-2 and HSV-1 infections each elicit strong immunoglobulin G (IgG) antibody responses in vivo to both HSV-2 gB-2 and to HSV-1 gB-1 (1, 2, 3); these antibodies are detectable early in acute infections, attain high titers, and persist for many years (4, 2, 5). Cross-reactive polyclonal antibody responses predominate (4, 6, 7). This exhibition of the related antigenicity of HSV gB-2 and gB-1 is consistent with the marked overall conservation of their amino acid sequences (86% at the amino acid level). Perhaps in consequence, the structures of gB-1 and gB-2 have not been previously broadly elucidated with respect to specific antigenic domains recognizing human antibodies which are clinically useful, especially type-specific antigenic regions capable of distinguishing HSV-1 from HSV-2 infection.
While human IgG antibodies cross-reactive to whole or randomly lysed gB-2 and gB-1 are thus known, virus type-specific antibodies have only been reported in murine models. Immunization of laboratory animals with purified virion-derived gB-2 elicits antibodies that neutralize HSV-2 infectivity in vitro and protect susceptible animal hosts from experimental HSV-2 infections (reviewed in Seminars in Pediatric Infections 2: 178-185, Stanberry, 1991 and Microbiol. and Immunol. 179: 137-158, Burke, 1992).
HSV-2 infections and HSV-1 infections elicit strong IgG antibody responses to gB-2 and to gB-1, respectively. gB-2 responses include antibodies that cross-react strongly with native gB-1, and gB-1 responses include antibodies that cross-react strongly with native gB-2. Therefore, gB-2 reactivities and gB-1 reactivities do not differentiate between antibody responses to HSV-2 infections and antibody responses to HSV-1 infections. Immune responses to most HSV-2 proteins include antibodies that cross-react with the homologous HSV-1 protein, and vice versa. Linear epitopes that react with type-specific, virus-neutralizing murine monoclonal antibodies have been localized to the extracellular amino-terminal segment of gB-1 (8, 9, 10, 11, 12, 13, 14). However, little information has been available regarding the locations and type specificities of gB epitopes recognized by human HSV antibodies (8, 15, 14). Vaccines comprising both recombinant gB-2 and gB-2 are currently undergoing clinical trials (16, 17), but efficacy or type-specificity of gB-2 in this application is not yet known.
The above numerals refer to the following publications, all incorporated herein by reference:
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