Antibody, cytokine and cytotoxic T lymphocyte responses in chimpanzees immunized with human papillomavirus virus-like particles.

We evaluated antibody, cytokine (IFN-gamma, IL-5, TNF-alpha), and cytotoxic T lymphocyte (CTL) responses in chimpanzees immunized with monovalent or quadrivalent (HPV-6, -11, -16, -18) L1 virus-like particle (VLP) vaccines administered i.m. on aluminum hydroxyphosphate (alum) at weeks 0, 8 and 24. Maximum serum antibody titers to type-specific, neutralizing, conformational epitopes on HPV-11 or -16 L1 VLPs were detected by radioimmunoassay (RIA) four weeks after the second and third immunizations. HPV-11 and -16 neutralizing antibodies were also detected at similar time points with an Human papillomaviruses (HPV) neutralization assay using pseudovirions. Depending on the VLP type used for immunization, HPV type-specific cytokine responses were most frequently seen four weeks after the second or third immunizations and between weeks 44-52. Transient HPV-16 L1-specific CTL activity was observed only between weeks 16-24 in 3 of 22 (13.6%) chimpanzees immunized with HPV-16 L1 VLPs. These findings provide evidence that immunization with multivalent L1 VLPs on alum can evoke both neutralizing antibodies and Th1 and Th2 cytokine responses to several HPV types; however, induction of CTLs is infrequent.

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.

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.

Safety and immunogenicity of an HLA-based HIV envelope polyvalent synthetic peptide immunogen. DATRI 010 Study Group. Division of AIDS Treatment Research Initiative.

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 x 10(6)/l were enrolled. Eight patients received the C4-V3 PV immunogen emulsified in incomplete Freund's adjuvant in five intramuscular injections over 24 weeks, and two controls received incomplete Freund's 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-1(4489-5) 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.

Developmental regulation of lymphocyte-specific protein 1 (LSP1) expression in thymus during human T-cell maturation.

The lymphocyte specific protein 1 (LSP1) phosphoprotein is an F-actin binding molecule restricted to cells of hematopoietic origin in mice and humans. LSP1 is localized to the internal surface of the plasma membrane, the cytoplasm, and NP-40-insoluble actin filaments and is thought to mediate cytoskeleton-driven responses in activated leukocytes that involve receptor capping, cell-cell interactions and cell motility. Here, we generated two monoclonal antibodies (MAbs), 5E3 and 14G8, that are specific for human LSP1 to define the expression of LSP1 throughout human T-cell development. Both MAbs reacted with a 52-kDa protein in BW5147 cells transfected with human LSP1 cDNA in pcDNA3, but not in cells transfected with cDNA in an antisense orientation, indicating the specificity of 5E3 and 14G8 for human LSP1. In developing T cells, LSP1 was expressed on human fetal liver CD7+ NK and T-cell precursors, the CD7+, CD3-, CD4-, CD8- human stem cell line DU-528, and on CD4-, CD8- double-negative (DN) thymocytes. Immunohistochemistry and three-color flow cytometry analysis of fetal or postnatal thymocytes revealed that LSP1 was increasingly expressed during intrathymic human T-cell maturation. While immature CD4+CD8+ double-positive (DP) thymocytes expressed low to undetectable levels of LSP1, mature CD4+CD8- and CD4-CD8+ single-positive (SP) thymocytes expressed high levels of LSP1. Thus, LSP1 is developmentally regulated during T-cell maturation within the human thymus and may play a functional role in the motility of DN and SP thymocytes.

Intranasal immunization is superior to vaginal, gastric, or rectal immunization for the induction of systemic and mucosal anti-HIV antibody responses.

Vaginal anti-HIV antibody responses may be beneficial, and possibly required, for vaccine-induced protection against HIV infection acquired through receptive vaginal intercourse. We have previously determined that intranasal immunization with a hybrid HIV peptide and cholera toxin induced vaginal anti-HIV IgA responses in BALB/c and C57BL/6 mice. To determine if vaginal, gastric, or rectal boosting would enhance the induction of vaginal anti-HIV IgA responses over those observed with intranasal immunization only, C57BL/6 mice were intranasally immunized with the hybrid HIV peptide T1SP10MN(A) and cholera toxin (days 0 and 14) and boosted via the vaginal, gastric, or rectal route (days 7 and 28). Four intranasal immunizations was superior to all other immunizations evaluated for the induction of plasma anti-peptide IgG, vaginal anti-peptide IgG and IgA, and peptide-specific delayed-type hypersensitivity. In addition, intranasal priming with gastric boosting was associated with greatly elevated total serum IgE concentrations whereas intranasal immunization only was associated with only a modest increase in total serum IgE. These results suggest that intranasal immunization is a viable route of immunization for the induction of systemic and mucosal anti-HIV immune responses.

Intranasal immunization with CTL epitope peptides from HIV-1 or ovalbumin and the mucosal adjuvant cholera toxin induces peptide-specific CTLs and protection against tumor development in vivo.

To evaluate the ability of mucosal immunization protocols using peptide immunogens to induce CTL responses, BALB/c and C57BL/6 mice were immunized intranasally (i.n.) with peptides corresponding to a known CTL epitope in HIV-1 glycoprotein 120 or OVA, respectively, and the mucosal adjuvant cholera toxin (CT). Intranasal immunization of BALB/c mice with a 10- or 15-amino acid peptide corresponding to a CTL determinant in HIV-1 glycoprotein 120 and CT induced peptide-specific CTLs in spleen cells that persisted through 35 days after the last immunization. Intranasal immunization of C57BL/6 mice with the octameric OVA peptide and CT produced similar results with detectable peptide-specific CTL in both the cervical lymph node and spleen. To test whether CTL induced by i.n. immunization with OVA peptide and CT were functional in vivo, groups of C57BL/6 mice were injected with E.G7-OVA tumor cells that express the OVA protein and monitored for tumor growth. Animals immunized i.n. with OVA and CT were protected against tumor development as efficiently as animals immunized by the potent CTL induction protocol of i.v. injection with OVA-pulsed dendritic cells. Intranasal immunization with peptides corresponding to known CTL epitopes and CT provides a noninvasive route of immunization for the induction of CTL responses in vivo.

The V3 domain of SIVmac251 gp120 contains a linear neutralizing epitope.

Antisera to 21 synthetic peptides containing hydrophilic sequences of simian immunodeficiency virus strain mac251 (SIVmac251) gp120 and gp32 were tested for the ability to neutralize SIVmac251. Goat antisera raised to peptides SP-1 and SP-1V containing the carboxy-terminal portion of the V3 domain of SIVmac251 gp120 between amino acids 327 and 339 inhibited syncytium formation (90% inhibition at a 1/1024 dilution) and cell killing of CEMx174 cells by SIVmac251 (50%) inhibition of cell killing at a dilution of 1/5832), SIVDeltaB670 (1/568), and SIVsmH4 (1/740). Neutralizing antibodies to SIVmac251, SIVDeltaB670, and SIVsmH4 could be adsorbed by peptides containing a neutralizing V3 sequence of SIVmac251 gp120 (GLVFHSQPIND, amino acids 329-339) but not by peptides lacking this sequence. This V3 neutralizing region corresponds to a homologous V3 neutralizing site within HIV-2 gp120 reported by Björling et al. 1991, Proc. Natl. Acad. Sci. USA 88, 6082-6086, 1994, J. Immunol. 152, 1952-1959). Antibodies in 20 of 31 sera obtained from rhesus macaques infected with SIVmac251 reacted with a peptide containing the entire V3 sequence of SIVmac251 gp120, whereas no sera contained antibodies reacting with the V3 neutralizing site between amino acids 329 and 339. Low levels of antibody-mediated recognition and subsequent lack of selective pressure against this linear V3 neutralizing site might in part explain why this region is not a dominant neutralizing site and also why sequences within V3 do not vary during the course of SIV infection.

Mucosal immunity to HIV-1: systemic and vaginal antibody responses after intranasal immunization with the HIV-1 C4/V3 peptide T1SP10 MN(A).

To optimize mucosal immune responses to the HIV-1 peptide vaccine candidate T1SP10 MN(A), we intranasally immunized BALB/c and C57BL/6 mice with C4/V3 HIV-1 peptide together with the mucosal adjuvant cholera toxin (CT). Four doses over a 4-wk period resulted in peak serum anti-peptide IgG titers of > 1:160,000 in BALB/c mice and > 1:520,000 in C57BL/6 mice, and significant levels (>1:30,000) persisted in both strains of mice for longer than 6 mo. Furthermore, intranasal immunization with peptide and CT induced serum IgG reactivity to HIV-1 gp120 and HIV-1(MN) neutralizing responses. The primary anti-peptide IgG subclass was IgG1, suggesting a predominant Th2-type response. In addition to elevated serum anti-peptide A responses, intranasal immunization with T1SP10 MN(A) and CT induced both vaginal anti-peptide IgG and IgA responses, which persisted for 91 days in both strains of mice. Vaginal anti-HIV IgA was frequently associated with secretory component, suggesting transepithelial transport of IgA into vaginal secretions. Cervical lymph nodes contained the highest relative concentration of anti-T1SP10 MN(A) IgG-producing cells, while the spleen was the next major site of anti-T1SP10 MN(A) IgG-producing cells. Ag-specific proliferative responses were also detected in cervical lymph node and spleen cell populations after intranasal immunization with T1SP10 MN(A) and CT. In addition, intranasal immunization with T1SP10 MN(A) and CT was able to induce anti-HIV cell-mediated immunity in vivo as indicated by the induction of delayed-type hypersensitivity. Therefore, intranasal immunization with hybrid HIV peptides provides a noninvasive route of immunization that induces both long-lived systemic and mucosal Ab responses as well as cell-mediated immunity to HIV.

Characterization of an antigen shared by human thymic epithelium and human T cell leukemia virus p19 Gag protein.

The molecular basis for cross-reactive antibody binding to human T cell leukemia virus type I (HTLV-I) p19 core protein and human thymic epithelium has been defined with two monoclonal antibodies (mAbs), 12/1-2 and 13B12, raised to HTLV-I p19. The mAb 12/1-2 has previously been shown to react with HTLV-I p19, HTLV-II p22, and antigens of normal human thymic epithelium, placenta, and foreskin, whereas mAb 13B12 binds only to the carboxyl terminus of HTLV-I p19. In the present study, mAb 12/1-2 bound to a subset of Triton X-100-insoluble intermediate filaments in human thymic epithelium also recognized by antikeratin antibodies AE1 and AE3. The mAb 12/1-2 also reacted in Western blot assays with proteins of 54, 46, and 40 kDa present in extracts of human thymic epithelium and with hexameric peptides containing overlapping sequences of HTLV-I p19 with the amino acids IPP (amino acids 117-119). In contrast, the HTLV-I-specific mAb 13B12 did not bind to human thymic epithelium and reacted with a single hexameric peptide containing the carboxy-terminal HTLV-I p19 sequence IPPPYV (amino acids 117-122). Binding of mAb 12/1-2 to thymic epithelium could be inhibited by adsorption with peptide SP-79 containing a C-terminal sequence (amino acids 112-125) of p19. The crossreactive IPP site is within a region of p19 that has been previously shown to be highly immunogenic in HTLV-I-infected individuals and that is also encoded by genes or mRNA of human cytokeratin 17, keratin 4, epidermal cytokeratin 2, and 50-kDa type I epidermal keratin. Thus, our studies define the sequence of a cross-reactive antigen on HTLV-I p19 that is also associated with keratin intermediate filaments from human thymic epithelium and other normal human tissues and that could serve as a focus of an autoimmune response during HTLV-I infection.

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.

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 cell 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.

Synthetic peptide in mineral oil adjuvant elicits simian immunodeficiency virus-specific CD8+ cytotoxic T lymphocytes in rhesus monkeys.

In view of the importance of cell-associated virus in AIDS virus transmission, an HIV vaccine should be able to induce a virus-specific CTL response. Traditional subunit vaccines have not elicited virus-specific CD8+ MHC class I-restricted CTL. We have used the simian immunodeficiency virus of macaques (SIVmac)/rhesus monkey model to explore the use of CTL epitope peptide-helper peptide conjugates for the vaccine elicitation of AIDS virus-specific CTL. We found that both the CTL epitope peptide-helper peptide conjugate and the CTL epitope peptide alone, when delivered in an emulsion with IFA, induced CTL epitope-specific CD8+ MHC class I-restricted CTL. These effector cells recognized processed viral protein and were readily cloned from PBL of the immunized monkeys. Moreover, the cloned effector cells inhibited SIVmac replication in PBL. Immunization with the CTL epitope peptide used in this study also elicited a CD4+ PBL proliferative response, suggesting that the peptide also contained a helper epitope. These studies provide further evidence for the potential usefulness of peptide-based AIDS virus vaccines.

Mother-to-child transmission of human T-cell lymphotropic virus type I (HTLV-I) in Jamaica: association with antibodies to envelope glycoprotein (gp46) epitopes.

To study mother-to-child transmission of HTLV-I in Jamaica, we screened antenatal patients in Kingston, Jamaica, from 1983 to 1985. Of 2,329 women, 81 (3.5%) were HTLV-I seropositive. Two to three years later, 36 seropositive mothers were recontacted, and blood was drawn from them and their children. All sera were tested for HTLV-I antibodies, and mother's sera were additionally tested for HTLV-I whole-virus antibody titer, syncytium-inhibition neutralizing antibody titer, and titers to six synthetic peptides from the HTLV-I envelope glycoprotein gp46. Seventeen of 74 (23%) [95% confidence interval (CI) 15-34%] children were seropositive. HTLV-I transmission was associated with breast-feeding duration > 6 months [relative risk (RR) 3.2; CI 0.4-22.1], maternal age > 30 years (RR 2.8; CI 1.0-7.8), and higher maternal whole-virus antibody titer (RR 3.3; CI 1.3-8.5). After controlling for higher whole-virus antibody titer, transmission remained associated with higher titer of neutralizing antibody and higher titer of antibody to the peptide sp4a1, corresponding to amino acids 196-209 of the gp46 envelope glycoprotein. We conclude that mother-to-child transmission of HTLV-I in Jamaica is associated with longer duration of breast-feeding, older age, and higher HTLV-I antibody titer, in particular to a certain immunogenic portion of the gp46 envelope glycoprotein.

Induction of HIVMN neutralizing antibodies in primates using a prime-boost regimen of hybrid synthetic gp120 envelope peptides.

We have tested synthetic peptides composed of Th (T1) and V3 loop B cell neutralizing determinants [SP10 MN(A)] of HIVMN gp120 and the fusogenic (F) domain of gp41 as immunogens in rhesus monkeys. After two immunizations with either HIV env peptide T1-SP10 MN(A) or F-T1-SP10 MN(A), rhesus monkey serum neutralization titers against the HIVMN isolate ranged from 1:160 to 1:1400, and in cell-cell syncytium inhibition assay ranged from 1:20 to 1:80. However, in contrast to animals immunized with T1-SP10 MN(A), animals immunized twice with F-T1-SP10 MN(A) had no rise in anti-gp120 and neutralizing antibodies with an additional immunization with F-T1-SP10 MN(A) peptide. One of 4 rhesus monkeys (18987) had anti-HIVMN antibodies that cross-neutralized divergent HIV isolates HIVIIIB and HIVRF. Serum from animal 18987 neutralized 5 of 10 HIV isolates tested, and neutralizing activity against HIVIIIB of 18987 serum was absorbed with the conserved gp120 loop V3 sequence IGPGRAF. Anti-HIV neutralizing antibodies were boosted after a 6-mo rest by 500 micrograms of T1-SP10 MN(A) in 4 of 4 animals previously immunized with T1-SP10 MN(A) and in 2 of 2 animals previously immunized with F-T1-SP10 MN(A). However, immunization after 6-mo rest of animal 18987 with 500 micrograms of T1-SP10 MN(A) peptide, although boosting anti-HIVMN neutralizing antibodies, selectively did not boost cross-neutralizing anti-HIVIIIB antibodies. Thus, synthetic peptides containing T and B cell epitopes of HIV gp120 can induce high levels of anti-HIVMN neutralizing antibodies in primates.

The mitogenic activity of human T-cell leukemia virus type I is T-cell associated and requires the CD2/LFA-3 activation pathway.

The presence of a high number of activated T cells in the bloodstream and spontaneous proliferation of peripheral blood mononuclear cells in vitro are striking characteristics of human T-cell leukemia virus type I (HTLV-I) infection. The HTLV-I regulatory protein Tax and the envelope protein gp46 have been implicated in mediating the activation process. In this study, HTLV-I-producing cell lines and purified virus from the cell lines were examined for the ability to activate peripheral blood lymphocytes (PBLs) and Jurkat cells. Antisera and monoclonal antibodies against several cellular adhesion proteins involved in T-cell activation and against viral proteins were used to identify which molecules may be participating in the activation process. First, neither virus from a T-cell line, MT2, nor virus produced from the human osteosarcoma cell line HOS/PL was able to induce PBLs to proliferate. In contrast, both fixed and irradiated HTLV-I-producing T-cell lines induced proliferation of PBLs; HOS/PL cells did not activate PBLs. Second, HTLV-I-positive T-cell lines were capable of activating interleukin-2 mRNA expression in Jurkat cells. Induction of interleukin-2 expression was inhibited by anti-CD2 and anti-lymphocyte function-associated antigen 3 (LFA-3) monoclonal antibodies but not anti-human leukocyte antigen-DR, anti-CD4, anti-LFA-1, or anti-intercellular adhesion molecule 1. Similar results were obtained with PBLs as the responder cells. Furthermore, monoclonal antibodies and antisera against various regions of the HTLV-I envelope proteins gp46 and gp21 as well as p40tax did not block activation. These data indicate that HTLV-I viral particles are not intrinsically mitogenic and that infection of target T cells is not necessary for activation. Instead, the mitogenic activity is restricted to virus-producing T cells, requires cell-to-cell contact, and may be mediated through the LFA-3/CD2 activation pathway.

Conversion of an immunogenic human immunodeficiency virus (HIV) envelope synthetic peptide to a tolerogen in chimpanzees by the fusogenic domain of HIV gp41 envelope protein.

The fusogenic (F) domain of human immunodeficiency virus (HIV) gp41 envelope (env) protein has sequence similarities to many virus and mediates the fusion of HIV-infected cells. During a survey of the immunogenicity of HIV env peptides in chimpanzees, we have observed that HIV peptide immunogenicity was dramatically altered by the NH2-terminal synthesis of the gp41 F domain to an otherwise immunogenic peptide. We compared two hybrid peptide types comprised of T helper (Th) and B cell epitopes of HIV gp120 env protein for their immunogenicity in chimpanzees. The Th-B epitope hybrid peptides contained the HIV gp120 Th cell determinant, T1 (amino acids [aa] 428-440)-synthesized NH2 terminal to gp120 V3 loop peptides, which contain B cell epitopes that induce anti-HIV-neutralizing antibodies (SP10IIIB [aa 303-321] and SP10IIIB [A] [aa 303-327]). The F-Th-B peptide contained the HIV gp41 F domain of HIVIIIB gp41 (aa 519-530)-synthesized NH2 terminal to the Th-B peptide. Whereas Th-B peptides were potent immunogens for chimpanzee antibody and T cell-proliferative responses, the F-Th-B peptide induced lower anti-HIV gp120 T and B cell responses. Moreover, immunization of chimpanzees with F-Th-B peptide but not Th-B peptides induced a significant decrease in peripheral blood T lymphocytes (mean decrease during immunization, 52%; p < 0.02). Chimpanzees previously immunized with F-Th-B peptide did not respond well to immunization with Th-B peptide with T or B cell responses to HIV peptides, demonstrating that the F-Th-B peptide induced immune hyporesponsiveness to Th and B HIV gp120 env determinants. These observations raise the hypothesis that the HIV gp41 env F domain may be a biologically active immunoregulatory peptide in vivo, and by an as yet uncharacterized mechanism, promotes primate immune system hyporesponsiveness to otherwise immunogenic peptides.

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.

Mapping of homologous, amino-terminal neutralizing regions of human T-cell lymphotropic virus type I and II gp46 envelope glycoproteins.

Twelve synthetic peptides containing hydrophilic amino acid sequences of human T-cell lymphotropic virus type I (HTLV-I) envelope glycoprotein were coupled to tetanus toxoid and used to raise epitope-specific antisera in goats and rabbits. Low neutralizing antibody titers (1:10 to 1:20) raised in rabbits to peptides SP-2 (envelope amino acids [aa] 86 to 107), SP-3 (aa 176 to 189), and SP-4A (aa 190 to 209) as well as to combined peptide SP-3/4A (aa 176 to 209) were detected in the vesicular stomatitis virus-HTLV-I pseudotype assay. Higher-titered neutralizing antibody responses to HTLV-I (1:10 to 1:640) were detected with pseudotype and syncytium inhibition assays in four goats immunized with a combined inoculum containing peptides SP-2, SP-3, and SP-4A linked to tetanus toxoid. These neutralizing anti-HTLV-I antibodies were type specific in that they did not inhibit HTLV-II syncytium formation. Neutralizing antibodies in sera from three goats could be absorbed with peptide SP-2 (aa 86 to 107) as well as truncated peptides containing envelope aa 90 to 98, but not with equimolar amounts of peptides lacking envelope aa 90 to 98. To map critical amino acids that contributed to HTLV-I neutralization within aa 88 to 98, peptides in which each amino acid was sequentially replaced by alanine were synthesized. The resulting 11 synthetic peptides with single alanine substitutions were then used to absorb three neutralizing goat antipeptide antisera. Both asparagines at positions 93 and 95 were required for adsorption of neutralizing anti-HTLV-I antibodies from all three sera. Peptide DP-90, containing the homologous region of HTLV-II envelope glycoprotein (aa 82 to 97), elicited antipeptide neutralizing antibodies to HTLV-II in goats that were type specific. In further adsorption experiments, it was determined that amino acid differences between homologous HTLV-I and HTLV-II envelope sequences at HTLV-I aa 95 (N to Q) and 97 (G to L) determined the type specificity of these neutralizing sites. Thus, the amino-terminal regions of HTLV-I and -II gp46 contain homologous, linear, neutralizing determinants that are type specific.