Thomas J. Palker

Safety and immunogenicity of an HLA-based HIV envelope polyvalent synthetic peptide immunogen

Objective:To evaluate the safety and immunogenicity of a polyvalent (PV) HIV envelope synthetic peptide immunogen, C4-V3. The immunogen comprised four peptides containing T-helper epitopes from the fourth constant region (C4) of gp120 of HIV-1MN, and T-helper, cytotoxic T-lymphocyte HLA-B7-restricted, and B-cell neutralizing epitopes from the gp120 third variable region (V3) of four clade B HIV-1 isolates, HIV-1MN, HIV-1RF, HIV-1EV91, and HIV-1Can0A. Design:A pilot, Phase I controlled trial [Division of AIDS Treatment Research Initiative (DATRI) 010] conducted at a single center. Methods:Ten HIV-infected, HLA-B7-positive patients with CD4 cells > 500 × 106/l were enrolled. Eight patients received the C4-V3 PV immunogen emulsified in incomplete Freunds adjuvant in five intramuscular injections over 24 weeks, and two controls received incomplete Freunds adjuvant alone. All subjects were followed for 52 weeks. Results:Four out of eight C4-V3 PV recipients generated at least fourfold rise in serum antibody titers to at least three immunogen peptides in contrast to none of the control subjects. Four out of eight C4-V3 PV recipients and none of the controls had an at least fourfold rise in neutralizing antibodies to either HIV-1MN, HIV-1RF, or HIV-14489-5 laboratory-adapted HIV isolates. 3H-Thymidine incorporation assays of peripheral blood mononuclear cells increased at least fivefold over the baseline stimulation index to at least one of the immunogen peptides in two consecutive post-immunization timepoints in five out of eight C4-V3 PV recipients versus none of the controls. CD4 cell counts and plasma HIV RNA levels did not change in patients who received either C4-V3 PV or adjuvant alone. Adverse events consisted primarily of grade 1 injection site reactions in six subjects (four C4-V3 recipients, two controls). Conclusions:C4-V3 PV synthetic peptides demonstrated both immunogenicity and safety in HIV-infected patients.

Vaccine-Elicited V3 Loop-Specific Antibodies in Rhesus Monkeys and Control of a Simian-Human Immunodeficiency Virus Expressing a Primary Patient Human Immunodeficiency Virus Type 1 Isolate Envelope

ABSTRACT Vaccine-elicited antibodies specific for the third hypervariable domain of the surface gp120 of human immunodeficiency virus type 1 (HIV-1) (V3 loop) were assessed for their contribution to protection against infection in the simian-human immunodeficiency virus (SHIV)/rhesus monkey model. Peptide vaccine-elicited anti-V3 loop antibody responses were examined for their ability to contain replication of SHIV-89.6, a nonpathogenic SHIV expressing a primary patient isolate HIV-1 envelope, as well as SHIV-89.6P, a pathogenic variant of that virus. Low-titer neutralizing antibodies to SHIV-89.6 that provided partial protection against viremia following SHIV-89.6 infection were generated. A similarly low-titer neutralizing antibody response to SHIV-89.6P that did not contain viremia after infection with SHIV-89.6P was generated, but a trend toward protection against CD4+ T-lymphocyte loss was seen in these infected monkeys. These observations suggest that the V3 loop on some primary patient HIV-1 isolates may be a partially effective target for neutralizing antibodies induced by peptide immunogens.

Expression and immunogenicity of the entire human T cell leukaemia virus type I envelope protein produced in a baculovirus system

The entire envelope gene of human T cell leukaemia virus type I (HTLV-I) has been successfully expressed in a baculovirus non-fusion vector system. The HTLV-I envelope protein accumulated within the insect cells as inclusion bodies which allowed efficient recovery of the recombinant protein. In an attempt to study the role of the HTLV-I envelope glycoprotein as an immunogenic target, mice were immunized with the envelope protein inclusion bodies (env-I.B.) in the presence or absence of an adjuvant. Antibodies of broad specificity were produced against the HTLV-I envelope protein in the presence or absence of an adjuvant as detected by Western blotting, radioimmunoprecipitation and peptide ELISA. Neutralizing antibody was detected when env-I.B. immunizations were carried out in the presence of high doses of a new adjuvant composed of a mycobacterial cell wall extract. In a combined immunization regimen, env-I.B. were found to enhance and broaden the antibody response to the HTLV-I envelope glycoprotein, following priming with various recombinant vaccinia virus (RVV) constructs expressing either the entire native HTLV-I envelope (gp46 and gp21) or just the surface envelope protein (gp46). Increased titres of neutralizing antibodies were observed following priming with the RVV expressing gp46 only. Results indicate that immunization regimens that involve priming with RVV expressing HTLV-I envelope followed by boosting with recombinant baculoviral HTLV-I envelope might be useful in eliciting protective immune responses in vivo.

Characterization of the antibody response to three different versions of the HTLV-I envelope protein expressed by recombinant vaccinia viruses : induction of neutralizing antibody

Recombinant vaccinia viruses (RVV) designated RVV E1, RVV E2, and RVV E3, were constructed to express three different versions of the human T cell leukemia virus type I (HTLV-I) envelope proteins to determine which configuration elicits an optimal antibody response. RVV E1 expressed the native HTLV-I envelope proteins gp46 (surface protein) and gp21 (transmembrane protein), while RVV E2 expressed the envelope precursor with the proteolytic cleavage site deleted. The RVV E3 construct expressed only the external surface glycoprotein, gp46. Radioimmunoprecipitation and FACS analysis confirmed that the appropriate envelope proteins were expressed by RVV E1-, E2-, and E3-infected cells. Immunization studies were carried out using Balb/c, A/J, and C57BL/6 strains of mice. Balb/c mice responded poorly to immunization with all of the three RVV constructs. C57BL/6 mice produced neutralizing antibodies in response to immunization with all three constructs, whereas A/J mice developed neutralizing antibodies only when immunized with the RVV E1s construct. The results indicate that the humoral immune responses depend on the form of HTLV-I envelope proteins expressed by each RVV.

Design of Experimental Synthetic Peptide Immunogens for Prevention of HIV-1 and HTLV-I Retroviral Infections

The potential for using synthetic peptides to serve as components of protective vaccines or as immunotherapeutics (see Chapter 38) is being explored for a variety of viral or bacterial pathogens. Peptide immunogens that elicit some level of protection in animal models or in clinical trials have been described for foot-and-mouth disease virus (Bittle et al., 1982), murine mammary tumor virus (Dion et al., 1990), Venezuelan equine encephalitis virus (Hunt et al., 1990), respiratory syncytial virus (Trudel et al., 1991), Semliki Forest virus (Snijders et al., 1992), Sendai virus (Kast et al., 1991), lymphocytic choriomeningitis virus (M. Schulz et al., 1991), group A streptococci (Bessen and Fischetti, 1990), and Plasmodium falciparum (Rodriguez et al., 1990; Etlinger et al., 1991).

Comparative Analysis of the Antibody Response to the HTLV‐I gag and env Proteins in HTLV‐I Asymptomatic Carriers and HAM/TSP Patients: an Isotype and Subclass Analysis

We have compared the immunoglobulin isotype and IgG subclass and the titre of neutralizing antibody responses to the human T celt lymphotropic virus type I (HTLV‐I) between a group of asymptomatic HTLV‐I infected individuals and a group with the neurological disease HTLV‐I associated myelopathy/tropical spastic paraparesis (HAM/TSP). A western blot titration assay and an envelope peptide ELISA were used to determine the presence and titre of isotype and IgG subclass responses to the gag p19 and p24 proteins and to the envelope protein. Significant increases were observed in the number of individuals seropositive for a particular isotype and IgG subclass in the HAM/TSP group versus the asymptomatic group particularly for IgM and IgE and to a lesser extent, IgA. The predominant IgG subclasses to the HTLV‐I p19, p24 and envelope proteins were IgG1 and IgG3. This finding was also observed in the titres of the antibody responses to these HTLV‐I proteins. The HAM/TSP group also exhibited significantly higher neutralizing antibody titres than the asymptomatic group. This evidence suggests that some form of chronic immune stimulation might be involved in the immunopathogenesis of HAM/TSP. In addition, by following the Western blot titre to the IgM and IgE isotypes in particular, it may be possible to identify asymptomatic individuals progressing to HAM/TSP.

Comparison of the CD7 (3A1) Group of T Cell Workshop Antibodies

The CD7 group of Workshop antibodies react with a 40-Kd glycoprotein (gp40) on the surface of 85% of peripheral blood T cells, essentially all thymocytes, and leukemias of T cell precursors (1–5). Prototypical monoclonal antibodies for the CD7 group described by Haynes et al. (antibodies 3A1A and 3A1B) (1–3) and Morishima et al. (antibody 4A) (4) all identified a 40-Kd T lymphocyte antigen present on both T4+ and T8+ T cell subsets. Subsets of gp40+ T cells enhanced immunoglobulin (Ig) synthesis by B cells (1–4), suppressed B cell Ig synthesis when stimulated with Con A (1–3), responded optimally to PHA and Con A stimulation (1,2), and gave rise to alloreactive, cytotoxic T cells in allogeneic mixed leukocyte culture (4). Antibodies to gp40 did not induce [3H]thymidine incorporation in gp40+ T cells (1–4). Importantly, monoclonal antibodies to the gp40 T lymphocyte glycoprotein have been shown to be the most reliable diagnostic markers for the identification of T cell acute lymphoblastic leukemia (5,6).

Purification of envelope glycoproteins of human T cell lymphotropic virus type I (HTLV-I) by affinity chromatography.

The external envelope glycoprotein (gp46) and transmembrane glycoprotein (gp21) of human T-cell lymphotropic virus type I (HTLV-I) were isolated from lysates of HTLV-I-infected HUT-102 cells by affinity chromatography. Fifty ml aliquots of packed HUT-102 cells were extracted with 1% Triton X-100, and lysates were treated sequentially with an affinity column containing IgG from an HTLV-I+ human subject followed by chromatography of the bound fraction over a lentil lectin column. The identity of the purified envelope proteins was confirmed with a human monoclonal antibody (0.5 alpha) to gp46 and with rabbit antisera raised to a synthetic peptide from the C-terminus of gp21. Affinity-purified envelope glycoproteins were bound to microtiter wells and used in radioimmunoassay to detect murine and human anti-envelope antibodies to gp46 and gp21 molecules.

Human T-Cell Leukemia/Lymphoma Virus: Studies of Host-Virus Interaction

The human T-cell leukemia-lymphoma virus (HTLV) is a novel exogenous retrovirus (Poiesz et al. 1980, 1981; Gallo et al. 1982) recently found to be associated with adult T-cell leukemias in Japan (Yoshida et al. 1982; Haynes et al. 1982a), West Indies (Blattner et al. 1982; Catovsky et al. 1982), southeastern United States (Gallo et al. 1983a), Israel (Popovic et al. 1983), and other areas. Despite the diverse geographical clustering of HTLV-related leukemias and lymphomas, serological studies utilizing HTLV+ sera from patients with Japanese adult T-Cell leukemia (ATL), cutaneous T-cell lymphomas (CTCL), and T-cell lymphosarcoma cell leukemia (TLCL) have indicated that HTLV isolates from around the world are highly related (Popovic et al. 1982, 1983; Kalyanaraman et al. 1982a; Gallo et al. 1983a). Concordance between the primary structure of HTLV p24 predicted by sequence analysis of the HTLV provirus from Japanese ATL (Seiki et al. 1983) and the partial amino acid sequence of HTLV p24 from American ATL leukemia cells (Oroszlan et al. 1982), as well as nucleic acid homology (Wong-Staal et al. 1983) and shared proviral restriction enzyme cleavage sites between American and Japanese HTLV isolates, have prompted the adoption of terminology which defines the provirus of Japanese ATL as a strain (HTLVATL) of HTLV (Watanabe et al. 1983). In addition, a new type of HTLV designated as HTLV-II has been isolated from a patient (MO) with a T-cell variant of hairy cell leukemia (Kalyanaraman et al. 1982 b). Although the 24000-dalton core proteins of HTLV-I (also denoted here as HTLV) and HTLV-II are antigenically related, the restriction maps of HTLV-I and HTLV-II (MO) show few regions of overlap (Chen et al. 1983; Gelmann et al. 1984).

Effect of bismuth salts on systemic and mucosal immune responses to orally administered cholera toxin

While the antimicrobial and antisecretory effects of bismuth salts are well documented, little is known regarding their effects on immune responses to enterotoxins such as that of V. cholerae or to orally administered vaccine antigens. To evaluate the effects of Pepto Bismol (PB) on the induction of systemic and mucosal immune responses to cholera toxin (CT), C57BL/6 mice were orally administered 10 micrograms CT and PB, or mice were pretreated with PB 30 min prior to CT administration. When co-administered with CT, PB attenuated serum IgG1, IgG2a, IgG2b and IgG3 anti-CT responses in a dose-dependent manner and also reduced levels of circulating anti-CT IgA and total serum IgE. Similarly, anti-CT intestinal IgA responses were also decreased. However, when administered 30 min prior to CT, PB had little to no effect on serum or intestinal anti-CT immunoglobulin responses. Administration of bismuth subsalicylate (BSS), the active component of PB, or sodium salicylate did not reduce immune responses to CT, suggesting that the combination of BSS plus other constituents contained within PB contributed to the decreased immune response to CT. Moreover, bismuth subgallate alone inhibited antibody responses to CT. Our data are consistent with the hypothesis that, when administered orally with CT, PB and bismuth subgallate create a physical barrier to antigen uptake.