Long-term protection of chimpanzees against high-dose HIV-1 challenge induced by immunization.

A combination AIDS vaccine approach consisting of priming with adenovirus-HIV-1MN gp160 recombinants followed by boosting with HIV-1SF2 gp120 was evaluated in chimpanzees. Long-lasting protection, requiring only three immunizations, was achieved against a low-dose challenge with the SF2 strain of HIV-1 and a subsequent high-dose SF2 challenge administered 1 year later without an intervening boost. Notably, neutralizing antibody responses against both clinical and laboratory isolates developed in three chimpanzees and persisted until the time of high-dose challenge. The possibility that cytotoxic T-lymphocytes contribute to low-dose protection of a chimpanzee lacking neutralizing antibodies is suggested. Our results validate the live vector priming/subunit booster approach and should stimulate interest in assessing this combination vaccine approach in humans.

Role of virion-associated glycosylphosphatidylinositol-linked proteins CD55 and CD59 in complement resistance of cell line-derived and primary isolates of HIV-1.

This study investigates whether cell-derived glycosylphosphatidylinositol-linked complement control proteins CD55 and CD59 can be incorporated into HIV-1 virions and contribute to complement resistance. Virus was prepared by transfection of cell lines with pNL4-3, and primary isolates of HIV-1 were derived from patients' PBMCs. Virus was tested for sensitivity to complement-mediated virolysis in the presence of anti-gp160 antibody. Viral preparations from JY33 cells, which lack CD55 and CD59, were highly sensitive to complement. HIV-1 preparations from H9 and U937 cells, which express low levels of CD55 and CD59, had intermediate to high sensitivity while other cell line-derived viruses and primary isolates of HIV-1 were resistant to complement-mediated virolysis. Although the primary isolates were not lysed, they activated complement as measured by binding to a complement receptor positive cell line. While the primary isolates were resistant to lysis in the presence of HIV-specific antibody, antibody to CD59 induced lysis. Likewise, antibody to CD55 and CD59 induced lysis of cell line-derived virus. Western blot analysis of purified virus showed bands corresponding to CD55 and CD59. Phosphatidylinositol-specific phospholipase C treatment of either cell line-derived or primary isolates of HIV-1 increased sensitivity to complement while incubation of sensitive virus with purified CD55 and CD59 increased resistance to complement. These results show that CD55 and CD59 are incorporated into HIV-1 particles and function to protect virions from complement-mediated destruction, and they are the first report of host cell proteins functioning in protection of HIV-1 from immune effector mechanisms.

Isolation of human T-cell leukemia virus in acquired immune deficiency syndrome (AIDS).

Several isolates of a human type-C retrovirus belonging to one group, known as human T-cell leukemia virus (HTLV), have previously been obtained from patients with adult T-cell leukemia or lymphoma. The T-cell tropism of HTLV and its prevalence in the Caribbean basin prompted a search for it in patients with the epidemic T-cell immune deficiency disorder known as AIDS. Peripheral blood lymphocytes from one patient in the United States and two in France were cultured with T-cell growth factor (TCGF) an shown to express HTLV antigens. Virus from the U.S. patient was isolated and characterized and shown to be related to HTLV subgroup I. The virus was also transmitted into normal human T cells from umbilical cord blood of a newborn. Whether or not HTLV-I or other retroviruses of this family with T-cell tropism cause AIDS, it is possible that patients from whom the virus can be isolated can also transmit it to others. If the target cell of AIDS is the mature T cell as suspected, the methods used in these studies may prove useful for the long-term growth of these cells and for the identification of antigens specific for the etiological agent of AIDS.

Optimized protocol for detection of native, full-length HIV-1 envelope on the surface of transfected cells.

AIMS: Designing therapeutics against the HIV envelope glycoprotein (Env) is only as accurate as the structure of the Env they are targeting. Conserving the structure of the Env trimer is crucial for proper experimental assessment of antibody binding and neutralization. However, Env is notably difficult to express by transfection of a recombinant Env plasmid. To increase surface expression, researchers commonly utilize c-tail mutants of the gp41 transmembrane glycoprotein of HIV-1, but mutations and deletions in this region can impact the overall conformation and stability of the Env trimer. Multiple studies have shown that while tail mutants have higher Env surface expression, they are easier to neutralize and have altered trimer conformations compared with wild-type Env found in vivo on infected cells. To assess and characterize native cell surface Env structures, we sought a protocol that could reliably detect wild-type Env surface expression by flow cytometry. METHODS AND RESULTS: By avoiding fetal bovine serum-based buffers, significantly increasing the amounts of transfected plasmid and Env-specific antibody and by selecting a bright, biotin + streptavidin-PE detection system, we were able to increase the surface expression of transfected Env protein. CONCLUSION: This protocol will allow for more precise assessment of antibody binding, epitope exposure, and Env structure, all of which will contribute to designing more effective vaccines and immunotherapeutics.

Characterization by serial deletion competition ELISAs of HIV-1 V3 loop epitopes recognized by monoclonal antibodies.

Characterization of the sites recognized by antibody on the V3 loop of the envelope glycoprotein gp120 of HIV-1 was done by competition ELISAs on a series of four mouse mAbs, a human mAb and a human Fab. The solid-phase antigen consisted of biotin-YNKRK-RIHIGPGRAFYTTKN, a sequence from the center of the V3 loop of gp120MN, applied to streptavidin-coated wells. Competing antigens were two series of peptides with the HIV-1MN sequence each serially deleted at either the N or C terminus but kept constant at the other terminus. For each series, the amino acid at the deleting end needed to give a minimum KD was identified. The epitope was defined as the sequence including both of the identified amino acids as terminal amino acids. For the six antibodies reported, the epitope length ranged from seven to 14 amino acids. Use of a cyclic peptide as competing fluid-phase antigen suggested the influence of conformational constraints on presumed "linear" epitopes. The operationally-defined epitope was longer than the contact residues in one of two instances in which the X-ray crystallographic structure had been determined. The longer estimates of epitope length in the current study based on competition ELISAs with serial deletions suggest that non-contact residues are significant both in epitope definition and in functional applications including immunogen design.

Response of mononuclear cells from HIV-infected patients to B-cell mitogens: correlation with immunological and clinical features of disease progression.

Proliferation of mononuclear cells from HIV-seropositive patients to B-cell mitogens was studied in the absence and presence of mixed lymphocyte culture supernatants (MLC-sup). The results show: (1) patients' responses to B-cell mitogens overlap with normal responses but are, on average, consistently lower than normal; (2) the addition of MLC-sup increases the proliferative responses to T-cell-independent mitogens, but does not bring patient's responses up to control levels; (3) HIV-positive patients in all clinical categories have decreased responses to B-cell mitogens. Although some patients with AIDS Centers for Disease Control (CDC) group IVC and IVD have the lowest responses, asymptomatic (CDC group II) and AIDS-related complex (ARC; CDC groups III/IVA and IVB) patients also show significant defects. (4) The same patients were recategorized using an immunological staging system. Those patients with more normal immunohematological parameters have significantly greater responses to mitogens compared with patients with more abnormal immunological parameters. The data suggest that immunological staging could provide more information than clinical classification with respect to the underlying immunopathogenic events occurring in HIV infection.

HIV phenotype correlates with the relative amounts of lymphocyte function-related molecule 1 (LFA-1) and major histocompatibility complex (MHC) class II in the virion envelope.

OBJECTIVE: The biological phenotype of HIV-1 has been associated with various aspects of its infectivity, including syncytium formation and coreceptor usage. Adhesion molecules, present on both the target cell and the virus, have also been shown to play a role in the infectious process. A possible correlation between the presence of adhesion molecules in the envelope of HIV-1 with the biological phenotype of the virus is examined. DESIGN: The envelopes of 56 isolates of HIV-1 of known biological phenotype were analyzed for the presence of lymphocyte function-related molecule 1 (LFA-1) and major histocompatibility complex (MHC) class II molecules. METHODS: The coreceptor usage of each isolate was determined in a GHOST cell or a U87.CD4 infectivity assay. The presence of LFA-1 and MHC class II in each virus envelope was then determined using a virus-binding enzyme-linked immunosorbent assay (ELISA). RESULTS: Viruses using the chemokine receptor CCR5 have relatively higher levels of MHC class II than LFA-1 in their envelopes compared with those using CXCR4. CONCLUSIONS: The finding that there is a differential incorporation of MHC class II and LFA-1 molecules by CXCR4- and CCR5-using viruses augments the list of properties contributing to the biological phenotype of HIV-1. This may explain, in part, how CXCR4-using viruses are able to bind to and infect a broader range of cell types than CCR5-using viruses, and why CXCR4-using viruses are associated with a more aggressive disease course.

Presentation of HIV V3 loop epitopes for enhanced antigenicity, immunogenicity and diagnostic potential.

OBJECTIVE: To evaluate the immunological properties of a panel of human mucin MUC1/HIV V3 loop chimeras. DESIGN: The immunodominant epitope of MUC1 (APDTR) was found to be structurally isomorphous with the tip of the principle neutralizing determinant (PND) of HIV-1 (MN) (GPGRA). A panel of 120 residue, six tandem repeat (TR) and 60 residue, three TR chimeric antigens were constructed in which the repeating MUC1 epitope is replaced by HIV-1 PND. Each 20 residue TR contains one PND epitope. The PND of HIV-1 is presented in the native beta-turn conformation at the crest of each repeating knob structure of the mucin-like protein. METHODS: The antigenicity of the chimeric antigens were compared using enzyme-linked immunosorbent assay (ELISA) and HIV-infected patient sera. Structural effects of antibody-antigen interactions were determined using surface plasmon resonance, with human monoclonal antibodies, chimeric antigens and the cyclic and linear V3 loops. Immunogenicity of three versus six TR was measured in mice. RESULTS: Nine residues of the HIV PND substituted into the mucin backbone were equivalent to the 36 residue cyclic V3 loop in ELISA. The 120 residue antigens induced high titer, immunoglobulin (Ig) M and IgG, and HIV-specific antibodies in mice. CONCLUSIONS: MUC1/V3 chimeras efficiently detect HIV-specific antibodies in patient sera. Multivalent presentation of the PND is advantageous for higher affinity antibody-antigen interactions and for inducing HIV-specific IgM and IgG antibodies.

Human monoclonal antibodies to the V3 loop of HIV-1 gp120 mediate variable and distinct effects on binding and viral neutralization by a human monoclonal antibody to the CD4 binding site.

Interactive effects between human monoclonal antibodies specific for the V3 loop (257-D and 447-D) and an epitope within the CD4 binding site (F105) of HIV-1 gp120 were evaluated for neutralization of viral cytopathogenicity and binding to HIV-infected cells. Regardless of antibody pair, only additive effects were observed in neutralization of MN and SF2 virus though each antibody alone had potent neutralizing activity on these strains. Significant cooperativity was observed between F105 and 447-D in neutralization of RF. Relatively high concentrations (> 100 micrograms/ml) of each individual antibody are required for partial neutralization (25--40%) of RF. Coincubation with 10 micrograms/ml of each antibody increased neutralization activity 3--4-fold more than predicted for additive effects alone. No enhancement was seen upon coincubation of F105 with 257-D which does not neutralize RF. Antibody interactions with native antigen on HIV-infected cells was measured by flow cytometry. Results were consistent with neutralization results in the majority of flow cytometry experiments; however, enhanced binding did not necessarily predict enhanced neutralization. These data support the notion that either a conformational change occurs with binding of V3 loop antibodies which enhances the binding and neutralizing activity of antibodies directed to the CD4 binding site of gp120 or vice versa, or new antigenic sites are exposed by the V3 loop antibodies on cell surfaces and virions. Of importance, cooperativity is observed even at very low antibody concentrations.

Studies with monoclonal antibodies to the V3 region of HIV-1 gp120 reveal limitations to the utility of solid-phase peptide binding assays.

Using human monoclonal antibodies (HuMAbs) r(1)-447 (L-736,523) and 19b to the V3 region of HIV-1 gp120, we have explored epitope presentation on V3-peptides and on the corresponding gp120 proteins. HuMAb r(1)-447 binds strongly to the MN and SF-2 peptides and gp120 proteins. In contrast, while this HuMAb binds equally avidly to both the HxB2 and the BRU/BH10 peptides, it binds but weakly to the HxB2 V3 loop on gp120 and fails to bind at all to BH10 gp120. Thus, the solid-phase peptide binding assay can falsely predict reactivity of an MAb with a gp120 protein. Conversely, HuMAb 19b fails to bind to a peptide from the V3 loop of HIV-1 AD-6 in solid-phase assays, but binds to the same peptide in solution and also to AD-6 gp120. Thus, the solid-phase peptide binding assay can fail to predict reactivity of an MAb with a gp120 protein. Furthermore, serum antibodies from individual AD-6 do not react well with the AD-6 V3-peptide in a solid-phase assay, but react strongly with the corresponding MN V3-peptide. On the basis of peptide binding assays, we had assumed that the AD-6 virus was "MN-like" with a prototypic North American/European subtype B GPGR motif at the crown of the V3 loop. However, direct sequencing demonstrates that the AD-6 V3 loop contains a variant GPGK motif. This highlights a limitation of V3-peptide-based assays for serotyping viruses.

Immunoconjugates containing ricin A chain and either human anti-gp41 or CD4 kill H9 cells infected with different isolates of HIV, but do not inhibit normal T or B cell function.

We have previously reported that immunoconjugates composed of deglycosylated ricin A chain coupled to either recombinant (r) CD4 or two different monoclonal human anti-gp41 antibodies (rCD4-dgA and anti-gp41-dgA, respectively) are specifically toxic to HIV-infected lines of human T cells (H9) and monocytes (U937). In order to further evaluate these immunoconjugates as potential therapeutic reagents for killing HIV-infected cells, H9 cells infected with five different isolates of HIV were used as target cells in vitro. All three HIV-specific immunoconjugates were toxic to H9 cells infected with each HIV isolate, but were virtually nontoxic to uninfected cells. Chloroquine markedly potentiated the specific toxicity of all three conjugates, particularly the anti-gp41-dgAs. None of the conjugates affected the ability of normal peripheral blood B cells to respond to mitogen or the ability of normal T cells to respond to alloantigens.

Immunosuppression and infection in multiple myeloma.

Patients with multiple myeloma are at increased risk of severe bacterial infection. A variety of immune deficits has been described in such patients, including a decreased primary antibody response and defects in complement and granulocyte function. The depressed humoral response appears to result primarily from the activity of suppressor monocytes. Pneumovax (Merck Sharp & Dohme, West Point, Penn) should be administered to patients with myeloma, although its effectiveness in this population has not been proven. The role of other potential modalities of treatment and prophylaxis, such as IV gamma globulin, requires further study.

A disposable diffusion chamber system used to study immunoregulation by soluble factors.

A disposable two-chamber culture system is described which is useful for studying various soluble factors which regulate the immune response. This method requires small numbers of cells and less than 2 ml of medium per culture. The vessel is compact, relatively inexpensive and all component parts are readily available from commercial suppliers. This culture system circumvents the major problems associated with dual chamber methods previously developed for the study of immunoregulatory molecules affecting antibody production.

An immuno-dot blot assay for the detection of antibody to HIV.

This paper describes a simple and rapid immuno-dot blot assay for the detection of antibody to HIV. It utilizes nanogram quantities of HIV lysate, immobilized on nitrocellulose paper, and microliter quantities of serum or culture supernatants for the detection of antibody. The test is highly sensitive and specific. It offers the opportunity to perform multiple assays at a minimal cost and provides the additional advantage of not requiring any sophisticated or expensive equipment to perform the test or interpret the results.

Culture and recovery of macrophages and cell lines from tissue culture-treated and -untreated plastic dishes.

Macrophages can be separated from other cell types by their ability to readily attach and spread on glass or on plastic surfaces which are treated for optimal growth of cultured cells (tissue culture-treated plastic). To detach macrophages from these surfaces, techniques must be used which require prior preparation of special flasks or vessels, utilize expensive equipment, are time-consuming and almost uniformly require that the macrophages be exposed to various chemicals. We now report that macrophages can be enriched and recovered efficiently after attachment to disposable polystyrene bacteriologic petri dishes simply by gentle scraping with a rubber policeman. In this paper we compare this method to others currently in use in which resident peritoneal cells, peritoneal exudate cells or cells from bone marrow-derived cultures are detached from treated dishes using cold shock, chelating agents and lidocaine. In all studies, advantages were noted when cells were incubated in untreated dishes and detached by gentle scraping. In addition, untreated dishes supported the growth of adherent cell lines IC-21 and L929B and yielded large numbers of cells, with high viability, which were easily harvested.

Association of human immunodeficiency virus infection and autoimmune phenomena.

Patients infected with human immunodeficiency virus have a variety of presentations including fevers, lymphadenopathy, rash, renal dysfunction, and neurologic and hematologic disorders. Many of these features are also seen in patients with systemic lupus erythematosus (SLE). Herein are described five patients ultimately diagnosed as having acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC) in whom the differential diagnosis included SLE because of multi-system disease and autoimmune phenomena, especially positive antinuclear antibodies. Serum samples from 151 consecutive patients with AIDS or ARC were examined and 19 with low titer-positive antinuclear antibodies were found (17 at 1:20 and two at 1:160). These observations suggest that SLE and human immunodeficiency virus infection may share clinical and serologic features.

Immunologic abnormalities in homosexual men. Relationship to Kaposi's sarcoma.

Studies were performed to define the immunologic status of various groups of homosexual men including homosexual men with Kaposi's sarcoma, healthy homosexual men who were of similar ages to the homosexual patients with Kaposi's sarcoma and homosexual men with hyperplastic lymphadenopathy. Heterosexual men with Kaposi's sarcoma were also studied. Immunologic parameters which were examined included serum immunoglobulin levels, enumeration of B cells, T cells, and T-cell subsets, and quantitation of lymphocyte responsive to phytohemagglutinin (PHA) and pokeweed mitogen (PWM). Significant immunologic abnormalities were observed in all three groups of homosexuals studied. These were most severe in the homosexuals with Kaposi's sarcoma, somewhat less severe in homosexual men with lymphadenopathy, and least marked but still significant in healthy homosexual men. Heterosexual men with Kaposi's sarcoma displayed essentially normal immunologic profiles. The possible etiologic factors underlying the immunologic abnormalities in the male homosexual population studied and the role of an altered immune system in the development of and the fulminant course of Kaposi's sarcoma in these patients are discussed.

Mechanisms contributing to the neutralization of HIV-1.

The phenomenon of virus neutralization is a function of three variables: the antibody (Ab), the virus and the target cell. Variation in any one of these parameters may drastically affect the results of assays for neutralization. In focusing on the virus as a variable in assays for the neutralization of HIV-1, it has been shown that the range of sensitivity of primary HIV-1 isolates is quite large. This may be due to the structure and biology of the virus particle, its density of envelope proteins, its ability to retain or shed these proteins, and its phenotype, determining the type of cells it will infect. The Ab used for neutralization also contributes to the efficiency of neutralization. Thus, the 'match' between the specificity of the Ab and the structure and availability of the epitope on the virus will affect the interaction and contribute to the resultant reduction in virus infectivity. Similarly, the strength of interaction between the virus and the neutralizing Ab, dependent on the affinity of the Ab for the virus epitope, will also be a determining factor. However, other factors contribute to the neutralization sensitivity of primary isolates of HIV-1. One factor that has been almost completely overlooked in the recent literature is the role that the target cell plays in revealing reduced virus infectivity. The facility and mechanism through which different cell types bind a virion and are infected by it will contribute profoundly to the efficiency with which Ab-mediated neutralization can be detected. These factors are discussed below with particular reference to interpreting (and re-interpreting) the current literature on HIV-1 neutralization.

The implications of antigenic diversity for vaccine development.

The reactivity of human monoclonal and polyclonal anti-HIV-1 antibodies demonstrates that shared epitopes, including those that induce neutralizing antibodies, exist and are recognized by the human immune system. A priori, there is no reason why cross-clade neutralizing antibodies could not be induced by an appropriately constructed HIV vaccine. But to construct such a vaccine, it is critical to understand, as completely as possible, the antigenic structure of HIV, to establish and identify immunologic classifications for HIV, and to choose rationally the minimum number and types of viruses from these immunologic groupings that will induce the broadest protective responses.

Characterization of human monoclonal antibodies selected with a hypervariable loop-deleted recombinant HIV-1(IIIB) gp120.

Recombinant gp120 of the HIV-1(IIIB) isolate (BH10 clone) has been mutated to form the PR12 protein with the first 74 C-terminal amino acids and the V1, V2 and V3 hypervariable loops deleted. A variety of studies have shown that the CD4 binding domain (CD4bd) is very well exposed in PR12 in contrast to rgp120(LAI). Using PR12 for selection of human monoclonal antibodies (MAbs) from HIV-infected individuals, five MAbs were generated with specificities to the epitopes overlapping the CD4bd (1570A,1570C,1570D,1595 and 1599). The three MAbs, 1570A, C and D, generated from one HIV-infected individual, represent one MAb as determined by sequence analysis of the V(H)3 region. Since the epitopes overlapping the CD4bd exhibit variability among HIV-1 clades, the specificity of anti-CD4bd MAbs were distinguished by differing patterns of binding to recombinant envelope proteins derived from clade A, B, C, D and E viruses. The PR12-selected MAbs were also compared with a panel of gp120-selected anti-CD4bd MAbs and showed a different range of specificities. MAb 1599 is clade B specific, MAb 1595 reacts with the A, B and D clades, while MAb 1570 recognises the most conserved epitope, as it binds to all proteins. The results show that the exposure of different epitopes in the CD4bd of the PR12 protein allows this protein to serve as an immunogen and to induce anti-CD4bd antibodies.