HIV-1 vaccine development: constrained peptide immunogens show improved binding to the anti-HIV-1 gp41 MAb.

The human immunodeficiency virus type I (HIV-1) transmembrane glycoprotein gp41 mediates viral entry through fusion of the target cellular and viral membranes. A segment of gp41 containing the sequence Glu-Leu-Asp-Lys-Trp-Ala has previously been identified as the epitope of the HIV-1 neutralizing human monoclonal antibody 2F5 (MAb 2F5). The 2F5 epitope is highly conserved among HIV-1 envelope glycoproteins. Antibodies directed at the 2F5 epitope have neutralizing effects on a broad range of laboratory-adapted HIV-1 variants and primary isolates. Recently, a crystal structure of the epitope bound to the Fab fragment of MAb 2F5 has shown that the 2F5 peptide adopts a beta-turn conformation [Pai, E. F., Klein, M. H., Chong, P., and Pedyczak, A. (2000) World Intellectual Property Organization Patent WO-00/61618]. We have designed cyclic peptides to adopt beta-turn conformations by the incorporation of a side-chain to side-chain lactam bridge between the i and i + 4 residues containing the Asp-Lys-Trp segment. Synthesis of extended, nonconstrained peptides encompassing the 2F5 epitope revealed that the 13 amino acid sequence, Glu-Leu-Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn, maximized MAb 2F5 binding. Constrained analogues of this sequence were explored to optimize 2F5 binding affinity. The solution conformations of the constrained peptides have been characterized by NMR spectroscopy and molecular modeling techniques. The results presented here demonstrate that both inclusion of the lactam constraint and extension of the 2F5 segment are necessary to elicit optimal antibody binding activity. The ability of these peptide immunogens to stimulate a high titer, peptide-specific immune response incapable of viral neutralization is discussed in regard to developing an HIV-1 vaccine designed to elicit a 2F5-like immune response.

Purification of virus-like particles of recombinant human papillomavirus type 11 major capsid protein L1 from Saccharomyces cerevisiae.

Recombinant major capsid protein, L1 (M(r) = 55,000), of human papillomavirus type 11 was expressed intracellularly at high levels in a galactose-inducible Saccharomyces cerevisiae expression system by an HPV6/11 hybrid gene. The capsid protein self-assembled into virus-like particles (VLPs) and accounted for 15% of the total soluble protein. A purification process was developed that consisted of two main steps: microfiltration and cation-exchange chromatography. The purified VLPs were 98% homogeneous, and the overall purification yield was 10%. The final product was characterized by several analytical methods and was highly immunogenic in mice.

Human papillomavirus type 11 (HPV-11) neutralizing antibodies in the serum and genital mucosal secretions of African green monkeys immunized with HPV-11 virus-like particles expressed in yeast.

It has been shown previously that immunization of animals with recombinant virus-like particles (VLPs) consisting of the viral capsid proteins L1 or L1 plus L2 protected animals against experimental viral challenge. However, none of these experimental models addresses the issue of whether systemic immunization with VLPs elicits a neutralizing antibody response in the genital mucosa. Such a response may be necessary to protect the uterine cervix against infection with genital human papillomavirus (HPV) types. African green monkeys systemically immunized with HPV-11 VLPs expressed in Saccharomyces cerevisiae and formulated on aluminum adjuvant elicited high-titered HPV-11 VLP-specific serum antibody responses. Sera from these immunized monkeys neutralized HPV-11 in the athymic mouse xenograft system. Significant levels of HPV-11-neutralizing antibodies also were observed in cervicovaginal secretions. These findings suggest that protection against HPV infection of the uterine cervix may be possible through systemic immunization with HPV VLPs.

Characterization and evaluation of a recombinant hepatitis B vaccine expressed in yeast defective for N-linked hyperglycosylation.

The hepatitis B (HB) virus preS2 + 2 polypeptide (the M or middle envelope polypeptide) is N-glycosylated at the N4 residue of the preS2 domain when expressed in recombinant yeast. Hyperglycosylation at this amino acid residue (the addition of a large number of mannose residues to the core oligosaccharide), which occurs in common yeast strains, results in an HB vaccine with diminished immunogenicity. Hyperglycosylation can be prevented by expressing the preS2 + S polypeptide in mutant yeast strains (e.g. mnn9) which limit N-linked glycosylation to the addition of only core saccharide residues. An HB vaccine prepared from recombinant yeast expressing the non-hyperglycosylated preS2 + 2 polypeptide was of similar immunogenicity in mice to a licensed HB vaccine and was much more immunogenic in humans than the hyperglycosylated preS2 + 2 vaccine.

In vitro T cell immune responses to the preS2 antigen of the hepatitis B virus envelope protein in preS2 + S vaccine recipients. Absence of cross-reactivity of subtypes at a major T cell recognition site.

The immune responses to hepatitis B virus envelope antigen were investigated in 16 vaccine recipients after immunization with a recombinant yeast-derived preS2 + S (adw) vaccine for hepatitis B virus. After the completion of the three-slot immunization series, all vaccine recipients developed antibody to the S domain and anti-preS2 antibody. In vitro proliferative responses to preS2 (120-174) peptide were demonstrated in 10 of 16 vaccine recipients. Although reactivity could be demonstrated through the length of the preS2 peptide, the principal site of proliferative activity was contained within the preS2 (146-165) region of the peptide. The principal T cell reactive site coincides with a region of significant amino acid variability of the different hepatitis B virus serotypes. Cross-reactivity with a serotype (ayw) not present in the preS2 + S vaccine could not be demonstrated at this widely recognized T cell epitope. The low level of cross-reactivity demonstrated in a limited subset of the vaccine recipients was mediated through nondominant T cell reactive sites contained in the relatively conserved preS2 (120-146) region of the molecule. The identification of widely recognized but serotype-specific T cell epitopes in the preS2 region of the hepatitis B virus envelope antigen may be an important consideration in future vaccine development.

Detection of antibody to the PreS2 sequence of the hepatitis B virus envelope protein using an immuno-ligand assay with a silicon sensor detection system.

Sensitive immunoassays are essential for establishing the efficacy of recombinant vaccines to hepatitis B virus (HBV). These experimental vaccines include the PreS2 and S domains of the HBV envelope protein. To facilitate measurement of antibody against HBV PreS2, we employed the immuno-ligand assay with silicon sensor-based detection. Labeling of immune reagents with the haptens biotin and fluorescein allows adaptation to the immunofiltration light addressable potentiometric sensor (LAPS) system. A biotinylated monoclonal anti-PreS2 antibody and anti-PreS2 in clinical serum samples competitively bind in liquid phase to a fluorescein labeled PreS2 + S antigen. Streptavidin mediates the immobilization on biotinylated nitrocellulose membranes. Fluorescein mediates binding of an anti-fluorescein urease conjugate to the immune complex. Urease serves as the signal-generating component which subsequently is measured in the LAPS reader. In comparison to a competitive RIA, the immuno-ligand assay demonstrated a four-fold improved sensitivity using a smaller sample volume. The higher sensitivity resulted in earlier detection of seroconversion during a clinical vaccine study.

Anti-PreS2 antibody assay for evaluating immune responses among recipients of recombinant hepatitis B PreS2 + S vaccine.

A competitive radioimmunoassay was developed for measuring hepatitis B virus (HBV) anti-PreS2 antibody. The assay has been demonstrated to be highly specific for anti-PreS2 antibody and not subject to interference by other antibodies to HBV-specific antigens. This assay was used to evaluate anti-PreS2 antibody responses in a hepatitis B PreS2 + S vaccine human clinical trial in healthy adults.

Isolation of multiple biologically and chemically diverse species of epidermal growth factor.

We have analyzed several lots of epidermal growth factor (EGF) purified from murine submaxillary glands including "receptor grade" EGF from Collaborative Research and EGF from Boehringer Mannheim Biochemicals. New England Nuclear uses "receptor grade" EGF to produce 125I-labeled EGF. Though these reagents are reported to be homogeneous, we found them to be a mixture of six species. A method was developed to separate this mixture into its component parts. The individual components were chemically characterized and tested for biological potency. N-terminal sequence analysis of the unfractionated EGF-mixture reveals three different sequences starting with residues 1, 2, or 3 of the mature peptide. Each component exhibited different degrees of mitogenic and EGF receptor binding activity indicating that the N-terminal region contributes to the biological response. The species representing the complete EGF peptide is the most active species in all biological assays. A rapid method for purification of homogeneous complete EGF from commercial EGF preparations is described.

Viral enhancement and interference induced in cell culture by hepatitis A virus: application to quantitative assays for hepatitis A virus.

Hepatitis A virus (HAV) growing in human diploid lung fibroblast (MRC5) monolayers can either interfere with or enhance the cytopathic effect of Newcastle Disease virus (NDV) challenge. Enhancement of NDV occurred if HAV-infected monolayers were challenged with a low multiplicity of infection of NDV and incubated at 35 degrees C. Interference occurred if HAV-infected monolayers were given a high NDV multiplicity of infection and incubated at 32 degrees C. These phenomena were applied to assays for quantifying HAV and may be useful in providing new insights into viral interference and enhancement.

Automated cytopathic effect (CPE) assays.

An automated CPE procedure has been developed that increases the precision and ease of performing titrations of measles, mumps and rubella viruses in vaccine materials. By this procedure, additions of cell suspensions and reagents and the dilution of samples are performed automatically by a modified Dynatiter instrument, using 96-well microtitre plates. Cell monolayers are stained with carbolfuchsin dye to eliminate the need for microscopic examination. Finally, the trays are read in an optical scanner and the end points calculated automatically by a programmable calculator. The increased accuracy and precision attained by performing greater numbers of replicate assays at reasonable cost will be of particular value to vaccine manufacturers.