Linkage between the frequency of muscular weakness and loci that regulate immune responsiveness in murine experimental myasthenia gravis.

Mice immunized with acetylcholine receptor (AChR) purified from Torpedo californica form anti-AChR antibodies and often develop muscular weakness and flaccid paralysis closely resembling the human disease myasthenia gravis. This condition, termed experimental myasthenia gravis (EMG), is strain dependent in that the frequency of paralysis is much greater in some strains than in others. Differences in the frequency of EMG might result from differences in the immune system or the neuromuscular junction. In these studies, we have identified two loci, the major histocompatibility complex (H-2) region on chromosome 17 and the region that contains the structural genes for the constant region of immunoglobulin heavy chains (IgCH region) on chromosome 12, which significantly effect the probability with which a mouse immunized with T. californica AChR can be expected to become paralyzed. One genotype (H-2b, Ig-1b) correlated with high susceptibility to EMG in four strains with three dissimilar backgrounds. These studies demonstrate that susceptibility to EMG is a heritable trait determined by at least two distinct loci that are linked to regions of the mouse genome that regulate immune responsiveness.

Experimental myasthenia gravis. A murine system.

Mice from eight inbred strains were immunized with acetylcholine receptor (AChR) purified from Torpedo californica. All mice developed high concentrations of serum antibodies (10(-6) M) against the immunogen and approximately 80% possessed antibodies reactive with mouse nicotinic AChR. 33% of the mice immunized (n = 236) developed muscular weakness and flaccid paralysis. Behavioral, electrophysiological, and pharmacological similarities were found between the experimentally induced muscular weakness and the disease myasthenia gravis. Susceptibility to experimental myasthenia was found to be strain dependent in that the frequency of paralysis was much greater in some strains than others. The occurrence of muscular weakness and flaccid paralysis did not correlate with the concentration of antibodies reactive with T. californica or mouse AChR. Anti-receptor antibodies which increased the rate of AChR degradation on the mouse muscle cell line, BC3H-1, were found in the serum of both myasthenic and nonmyasthenic mice. 40% of the mice tested possessed antibodies reactive with antigenic determinants present on mouse receptor but not T. californica receptor. The occurrence of antibodies unique to mouse receptor did not correlate with myasthenia. Thus, myasthenia in the mouse does not occur simply as a consequence of the presence of antibodies directed against cell surface antigenic determinants of AChR. If anti-AChR antibodies are both necessary and sufficient for the induction of myasthenia, then these studies suggest that populations of a particular structure and/or specificity are required. It is anticipated that the mouse model of myasthenia gravis will permit the regulation of the anti-receptor immune response to be studied in detail.

Critical amino acids in the lymphocyte function-associated antigen-1 I domain mediate intercellular adhesion molecule 3 binding and immune function.

We have identified amino acid residues within the evolutionarily conserved I domain of the alpha-chain (CD11a) of the leukocyte integrin leukocyte function-associated antigen (LFA) 1 that are critical for intercellular adhesion molecule (ICAM) 3 (CD50) binding. ICAM-3, a ligand of LFA-1, is thought to mediate intercellular adhesion essential for the initiation of immune responses. Using a panel of human/murine I domain chimeras and point mutants, we observed that the Ile-Lys-Gly-Asn motif, located in the NH2-terminal part of the CD11a I domain, is required for ICAM-3 but not ICAM-1 binding. These findings demonstrate that the I domain of CD11a contains distinct functional subdomains for ligand specific binding. An aspartic acid located at position 137, which is essential to ICAM-1/LFA-1 interactions (Edwards, C.P., M. Champe, T. Gonzalez, M.E. Wessinger, S.A. Spencer, L.G. Presta, P.W. Berman, and S.C. Bodary. 1995. J. Biol. Chem. 270:12635-12640), was also critical for ICAM-3 binding, whereas Ser at position 139 did not effect ICAM-1 or ICAM-3 binding. A synthetic peptide containing the Ile-Lys-Gly-Asn motif inhibited ICAM-3-dependent adhesion and proliferation of T cells at micromolar concentrations, suggesting that this peptide interferes with immune recognition. These observations underscore the importance of ICAM-3 in leukocyte function, and may lead to development of a new category of immunosuppressive agents.

Topogenic analysis of the human immunodeficiency virus type 1 envelope glycoprotein, gp160, in microsomal membranes.

The orientation in cellular membranes of the 856 amino acid envelope glycoprotein precursor, gp160, of human immunodeficiency virus type 1 was investigated in vitro. Variants of the env gene were transcribed using the bacteriophage SP6 promoter, translated using a rabbit reticulocyte lysate, and translocated into canine pancreatic microsomal membranes. Immunoprecipitation studies of gp160 variants using antibodies specific for various gp160-derived polypeptides provided evidence that the external (cell surface) domain of gp160 begins at the mature amino terminus of the protein and continues through amino acid 665. A stop-transfer sequence (transmembrane domain) was identified in a hydrophobic region COOH-terminal to amino acid 665 and NH2-terminal to amino acid 732. Protease protection experiments demonstrated that gp160 possesses a single cytoplasmic domain COOH-terminal to residue 707. Membrane extraction studies using carbonate buffer provided evidence that the 29 amino acid hydrophobic domain (residues 512-541) of gp160 was unable to serve as a stop-transfer sequence. Finally, we propose that the cytoplasmic tail of gp160 forms a secondary association with the microsomal membranes.

Protection from genital herpes simplex virus type 2 infection by vaccination with cloned type 1 glycoprotein D.

Guinea pigs were vaccinated with truncated herpes simplex virus type-1 (HSV-1) glycoprotein D produced in the genetically engineered mammalian cell line gD10.2. Vaccinated animals formed antibodies that neutralized both HSV-1 and herpes simplex virus type 2 (HSV-2) in an in vitro neutralization assay. Vaccinated animals were challenged with HSV-2 by intravaginal infection. Animals that received the immunogen in Freund's complete adjuvant were completely protected from the clinical manifestations of genital HSV-2 infection. Animals that received the immunogen incorporated in alum adjuvants were partly protected from clinical disease; the infections that did develop were significantly less severe than those that occurred in control animals injected with adjuvant alone. The results demonstrate that immunization with a purified viral protein can provide significant protection against primary genital infection by HSV-2 in guinea pigs.

Detection of antibodies to herpes simplex virus with a continuous cell line expressing cloned glycoprotein D.

The gene for glycoprotein D of herpes simplex virus type 1 (HSV-1) was expressed in stable mammalian cell lines. Glycoprotein D produced in these cells has a number of antigenic determinants in common with the native glycoprotein. Cell lines expressing glycoprotein D were used in an enzyme-linked immunosorbent assay to detect human antibodies to glycoprotein D. This strategy should prove useful in determining the extent to which the immune response to HSV-1 is directed toward glycoprotein D.

Neutralization of the AIDS retrovirus by antibodies to a recombinant envelope glycoprotein.

Mammalian cell lines have been engineered to produce a secreted form of the AIDS retrovirus envelope glycoprotein. The recombinant protein has been isolated from growth-conditioned culture media and used to immunize animals. Antibodies directed against the recombinant molecule were found to react with the envelope glycoprotein produced in virus-infected cells. Furthermore, these antibodies were able to directly inactivate the AIDS retrovirus in a neutralization assay in vitro. The expression system reported here should provide sufficient quantities of the AIDS retrovirus envelope protein for biological and vaccination studies.

Comparison of the immune response to recombinant gp120 in humans and chimpanzees.

OBJECTIVE: To assess similarities and differences in antibody responses to recombinant (r) HIV-1IIIB gp120 in chimpanzees, previously protected from HIV-1 infection, and human volunteers immunized in connection with a Phase I clinical trial. METHODS: Frozen sera from humans immunized with rgp120 from HIV-1IIIB and chimpanzees immunized with the same antigen or recombinant soluble gp160 were compared in a variety of serologic assays. RESULTS: The magnitude of the antibody response to gp120 was similar in both species; however, the half-life of the antibody response to rgp120 was approximately 4.5 times longer in humans (9 weeks) than in chimpanzees (2 weeks). Antibodies to gp120 in both species were broadly cross-reactive with gp120 from diverse isolates of HIV-1 and were effective in blocking the binding of gp120 to CD4. Antibody binding to native gp120 was greater than to denatured gp120 in both species. Antibody responses to the principal neutralizing determinant (V3 domain) and virus neutralization titers were approximately 10-fold lower in humans than chimpanzees. The relative avidity of antibody binding to gp120 was higher in the sera from the immunized chimpanzees than in the immunized humans. CONCLUSIONS: While the antibody responses to rgp120 elicited in man and chimpanzees were in many ways similar, significant differences did occur. Predictions made on the basis of chimpanzee immunogenicity studies over-estimated the potency of the virus neutralizing titers and under-estimated the duration of the antibody response achieved in humans.

Development of bivalent rgp120 vaccines to prevent HIV type 1 infection.

A new generation of "bivalent" gp120-based vaccines, effective against subtype B and subtype E viruses, has been developed. Antisera from rabbits and humans immunized with these vaccines are able to neutralize macrophage tropic and T-cell tropic viruses grown in activated peripheral blood mononuclear cells (PBMCs). These vaccines are now available for efficacy trials to determine the role of humoral immunity in providing protection against human immunodeficiency virus type 1 (HIV-1) infection.

Adhesion mediated by intercellular adhesion molecule 1 attenuates the potency of antibodies that block HIV-1 gp160-dependent syncytium formation.

Several lines of evidence suggest that leukocyte adhesion molecules can promote HIV-1-mediated cell fusion and syncytium formation. In the present studies, the human kidney cell line, 293, was transfected with the envelope glycoprotein gene of the MN strain of HIV-1 alone or cotransfected with a cDNA encoding intercellular adhesion molecule 1 (ICAM-1). It was found that 293 cells transfected with the HIV-1MN env gene expressed the HIV-1 polyglycoprotein precursor, gp160, and the mature gp120-gp41 complex. When mixed with a CD4+ T cell line (CEM), the gp160-transfected cells mediated heterotypic cell fusion and formed multinucleate syncytia. Virus-neutralizing monoclonal antibodies to the V2 and V3 domains of gp120 were able to inhibit syncytium formation, as were monoclonal antibodies to CD4. When ICAM-1 was coexpressed with gp160, syncytium formation between the transfected kidney cells and uninfected CD$+ T cells was markedly enhanced. Inhibitors of HIV-1 infectivity (e.g., monoclonal antibodies to gp120, recombinant soluble CD4) were able to prevent syncytium formation; however, the syncytium-blocking activity of these agents was significantly attenuated in cultures in which ICAM-1 was cotransfected with gp160. These results confirm that leukocyte adhesion molecules can promote gp160-mediated syncytium formation and demonstrate, for the first time, that adhesive interactions mediated by ICAM-1 and its contrareceptor, LFA-1, attenuate the syncytium-inhibiting activity of virus-neutralizing monoclonal antibodies and soluble CD4. These findings suggest that the type and magnitude of leukocyte adhesion molecules expressed on cells may be a significant variable in in vitro HIV-1 neutralization assays.

Recombinant HIV structural proteins detect specific cellular immunity in vitro in infected individuals.

Peripheral blood mononuclear cell (PBMC) cultures were established from patients with antibody to human immunodeficiency virus (HIV). Asymptomatically infected patients [5 of 19] had significant lymphocyte transformation responses induced in culture by a purified, recombinant envelope glycoprotein (rgp120) from the virus. A few (4 of 55) subjects with AIDS related complex (ARC) and no subjects with AIDS (0 of 29) had proliferative responses to this protein. These responses correlated directly with circulating levels of helper/inducer lymphocytes (p less than .01) and indirectly with virus antigen in blood (p = .04). Also, these responses occurred significantly less frequently than responses to herpes simplex virus (HSV) or cytomegalovirus (CMV) antigens in seropositive ARC patients (p less than .005). These data indicate that the frequency of immune cellular responses to rgp120 decline in association with disease progression, and become undetectable in frank AIDS. As rgp120-induced proliferation was not observed in cells from 15 seronegative immunocompetent subjects, this response appears immune specific. Immune T-lymphocyte-mediated responses to this HIV envelope glycoprotein may allow the prediction of future clinical events and may be useful in monitoring immune-enhancing therapy in patients with ARC and AIDS.

MN and IIIB recombinant glycoprotein 120 vaccine-induced binding antibodies to native envelope glycoprotein of human immunodeficiency virus type 1 primary isolates. National Institute of Allergy and Infectious Disease Aids Vaccine Evaluation Group.

The ability of antibody induced by MN and IIIB recombinant gp120 (rgp120) human immunodeficiency virus type 1 (HIV-1) vaccines to bind to oligomeric native HIV-1 envelope glycoproteins of primary isolates of HIV-1 was measured by flow cytometric indirect immunofluorescence assay (FIFA) in 25 uninfected, healthy adults. After three immunizations, MN rgp120 HIV-1 vaccine given alone and coadministered with IIIB rgp120 HIV-1 vaccine elicited antibody that bound to cells infected with each of a panel of six subtype B strains of HIV-1. Lower levels of vaccine-induced binding antibody were detected against envelope subtype A, D, and (EA) strains of HIV-1 than against subtype B strains. Priming immunization with IIIB rgp120 HIV-1 vaccine alone induced low levels of antibody capable of binding to envelope glycoprotein of primary isolate strains of HIV-1, and booster immunizations with MN rgp120 HIV-1 vaccine resulted in much higher antibody levels. We conclude that MN rgp120 HIV-1 vaccine was an effective inducer of antibody to native envelope glycoproteins of antigenically diverse primary isolates of HIV-1.

A sensitive radioimmunoprecipitation assay for the detection and quantitation of antibodies to the envelope glycoprotein gp120 of the human immunodeficiency virus (HIV-1).

A radioimmunoprecipitation (RIP) assay was developed to detect antibodies to the envelope glycoprotein gp120 of the human immunodeficiency virus (HIV-1). The assay, which utilized recombinant gp120 (rgp120), was quantitative, reproducible, and specific for antibodies to rgp120 or antibodies to native gp120 resulting from natural infection with HIV. Polyethylene glycol-8000 (PEG), used in the assay at a final concentration of 10% to precipitate immune complexes, was demonstrated to be effective in titering sera from different animal species. Provided samples were diluted at least 1:100, antibody titers could be determined either by the classical dilution method or by interpolation from a calibration curve prepared with a positive serum. The humoral response of animals immunized with rgp120 was monitored and a positive correlation was found between titers determined in the RIP assay and the ability of the sera to neutralize. In addition, RIP titers of HIV-positive human sera correlated very well with reactivity obtained in a commercial HIV immunoblot assay. The assay has the advantage of quantitation, fast turnaround time, and versatility.

Development of a single-shot subunit vaccine for HIV-1.

The successful development of an AIDS vaccine will require formulations that not only invoke the desired immunological response, but also are stable and easy to administer. A single shot MN rgp120 vaccine formulation comprised of MN rgp120 encapsulated in poly (lactic-coglycolic) acid (PLGA) microspheres was developed to provide an in vivo autoboost of antigen. These formulations were designed to yield an in vivo autoboost at 1, 2, 3 or 4-6 months. In addition, PLGA microspheres containing the adjuvant, QS21, were also prepared to provide an in vivo autoboost concomitant with antigen. In guinea pigs, these formulations yielded higher anti-MN rgp120 and anti-V3 loop antibody titers than alum formulations that were administered at higher antigen doses. Different doses of encapsulated MN rgp120 provided a clear and well-defined dose response curve for both anti-MN rgp120 and anti-V3 loop antibody titers. When soluble QS21 was mixed with the encapsulated MN rgp120, the antibody titers were increased by a factor of 5 over the titers with encapsulated MN rgp120 alone. An additional fivefold increase in antibody titers was observed for guinea pigs immunized with encapsulated MN rgp120 and QS21 on the same microspheres. These results suggest that the adjuvant properties of QS21 can be increased by microencapsulation in PLGA. Furthermore, antibodies induced by these preparations neutralized the MN strain of HIV-1. The neutralization titers for sera from animals immunized with MN rgp120-PLGA and soluble QS21 were greater than the titers obtained from guinea pigs that were treated with MN rgp120 and soluble QS21 at the same dose. Overall, these studies validate the in vivo autoboost concept, reveal a method for improving the adjuvant properties of QS21, and indicate the potential of future single shot vaccine formulations.

Genetic variants of HIV-1 in Thailand.

Serosurveys conducted prior to 1988 indicated a very low level of HIV-1 infection in Thailand, even among high-risk groups. The Ministry of Health has reported a dramatic increase in HIV-1 infection during the last three years. The geographic and demographic distribution of the epidemic is broad, involving multiple provinces and risk groups. Foci of higher incidence and prevalence have been noted in the urban center of Bangkok and in the northern provinces of Chiang Mai and Chiang Rai. Here we report the results of genetic characterization of 16 HIV-1 isolates from Thailand using a combination of polymerase chain reaction (PCR) typing and DNA sequencing. The complete sequence of gp160 (env) of five isolates, partial env sequence of six additional isolates, and the gag gene of two isolates were determined. Two highly distinct HIV-1 variants were found. One variant resembled those prevalent in North America and Europe; five of the isolates were of this type. The remaining eleven isolates were very similar to one another and represented a variant unlike any previously described. Phylogenetic tree analysis of complete env and gag genes placed the two variants on widely separated branches. Protein sequence comparisons indicate both general and specific features that distinguish the Northern Thailand variant both from the Bangkok variant and from virtually all previously sequenced HIV-1 isolates. A simple PCR test for distinguishing the two variants has been developed for use in epidemiologic surveys.

HIV-1MN recombinant glycoprotein 160 vaccine-induced cellular and humoral immunity boosted by HIV-1MN recombinant glycoprotein 120 vaccine. National Institute of Allergy and Infectious Diseases AIDS Vaccine Evaluation Group.

We evaluated prime-boost immunization with two recombinant envelope glycoprotein subunit vaccines (HIV-1MN recombinant gp160 vaccine in alum adjuvant [MN rgp160] and HIV-1MN recombinant gp120 vaccine in alum adjuvant [MN rgp120]) for safety and immunogenicity in healthy, HIV-1-uninfected adults. The rationale was to combine the helper T cell memory and binding antibody responses typically induced by rgp160 vaccines with the superior neutralizing antibody responses induced by rgp120 vaccines. In a double-blinded, controlled trial, volunteers were randomly assigned to receive MN rgp160 or adjuvant placebo, and a subset later received MN rgp120. The two vaccines were safe, but reactions to MN rgp160 and its adjuvant placebo exceeded those to MN rgp120. MN rgp160 induced IgG binding antibodies, including all IgG subclasses, to MN rgp160 in all vaccine recipients. HIV-1MN-neutralizing and anti-V3 MN peptide-binding antibodies were observed in a majority of volunteers after the fourth MN rgp160 immunization, but at lower levels compared with immunization with MN rgp120 in historical controls. HIV-1-binding, neutralizing, and fusion inhibition antibodies were boosted to the highest levels among MN rgp160 recipients after MN rgp120 booster injections. MN rgp120 boosting appeared to alter the distribution of MN rgp160 vaccine-induced, anti-MN rgp160 IgG subclass antibodies. MN rgp160 induced helper T cell memory, measured by lymphocyte proliferation, Thl and Th2 cytokine production, and skin testing. Strategies including both subunit vaccines may help maximize antibody and helper T cell memory responses to HIV-1 envelope glycoprotein.

Advancing AIDSVAX to phase 3. Safety, immunogenicity, and plans for phase 3.

AIDSVAX (VaxGen, Inc., South San Francisco, CA), a possible vaccine to protect against human immunodeficiency virus type 1 (HIV-1) infection, is being tested for efficacy in phase 3 studies. It has been tested for potential efficacy in chimpanzees, and tested for safety and immunogenicity in human clinical studies. Four candidate vaccines, each with a different envelope protein antigen or combination of antigens, have been produced in alum formulations. In both design and clinical testing, AIDSVAX has an excellent safety profile. Because these highly purified proteins were prepared using recombinant DNA technology, there is no possibility of these vaccines causing HIV infection. Having been administered to over 1200 people, the only side effects attributable to AIDSVAX have been local pain and inflammation at the injection site. After immunization, essentially all recipients developed a robust antibody response, including binding and neutralizing antibodies. The neutralizing antibodies peaked after a 12-month boost. Excellent memory is induced. Two phase 3 trials of two bivalent formulations will evaluate their efficacy. One trial will use a bivalent subtype B formulation. This trial in North America will involve 5000 men who have sex with men and heterosexual women at high risk. The other study will use a bivalent subtype B/subtype E formulation. This trial in Thailand and will involve 2500 intravenous drug users. Both studies will be randomized, double-blinded and placebo controlled. The volunteers will be followed for 3 years. The end points of the studies are infection, as defined by seroconversion to standard diagnostic tests, and viral load, as defined by commercial polymerase chain reaction (PCR) tests.

Factors affecting the susceptibility of different strains of mice to experimental myasthenia gravis.

Mice immunized with purified AChR (T. californica) invariably form anti-AChR antibodies and often develop a condition of extreme muscular weakness and flaccid paralysis. Pharmacological, physiological, and ultrastructural studies indicate that the pathophysiology of EMG in the mouse closely resembles that of human MG. The single episode of muscular weakness typically found in mouse EMG differs from the acute phase of rat EMG in that macrophages and other phagocytes do not appear to play an active role in the destruction of the neuromuscular junction. The frequency of paralysis in mice immunized with AChR is highly strain dependent and is not attributable to polymorphisms with respect to susceptibility to cholinergic blockade. The incidence of paralysis does not correlate with the magnitude of the humoral response to either T. californica or mouse AChR. Because both paralyzed and nonparalyzed mice form antibodies which are able to increase the rate of both junctional and extrajunctional AChR degradation, the mere presence of antibodies reactive with cell surface antigenic determinants of AChR is not sufficient for the induction of paralysis. While it is still possible that antibody-induced degradation of AChR may be necessary for the induction of paralysis, these studies rule out the possibility that antigenic modulation of AChR is sufficient to account for the induction of paralysis in mouse EMG. In the present studies alleles of the two loci were identified which significantly effect the probability with which mice immunized with AChR can be expected to become paralyzed, the MHC and the IgCH region. Because one genotype, H-2b, Ig-1b segregated with high susceptibility to EMG in four strains derived from three dissimilar backgrounds, these studies strongly suggest that susceptibility to the development of paralysis is a heritable trait determined by regions of the mouse genome which regulate immune responsiveness.

Development of a single-shot subunit vaccine for HIV-1. 2. Defining optimal autoboost characteristics to maximize the humoral immune response.

The design of a single-shot subunit vaccine for HIV-1 with polylactic-coglycolic acid (PLGA) sustained-release technology to effect an autoboost of antigen (MN gp120) at a given time after the primary immunization requires in-depth knowledge about the timing, the duration, and the need for coadjuvant in the autoboost. These questions cannot be answered unambiguously with PLGA microspheres, so we have conducted studies using Alzet minipumps to release antigen at prescribed times to mimic a PLGA autoboost. The results show that a discrete autoboost is preferred over continuous release of antigen, that the time profile of the autoboost (whether pulsatile or a 2-week continuous release) does not affect the booster immune response, and that only antigen is required in the booster immunization (a coadjuvant in the boost does not give higher titers).