Epitopes of the HIV-1-negative factor (nef) reactive with murine monoclonal antibodies and human HIV-1-positive sera.

Murine monoclonal antibodies (MAbs) raised against a recombinant nef protein fragment of human immunodeficiency virus type 1 (HIV-1) strain BH10 were characterized by an epitope mapping system using overlapping decapeptides. Four different immunogenic regions were identified. Ten human HIV-1-positive sera were tested in the same epitope mapping system, seven of these were reactive with four immunogenic regions. Two of the nef-specific epitopes recognized by human sera overlapped with the epitopes defined by the murine monoclonal antibodies. The reactivity of the monoclonal antibodies with the recombinant nef protein and with infected and uninfected cells were investigated in a variety of test systems. The results are discussed with respect to homologous regions of nef and cellular proteins.     

Recombinant peptides derived from the env-gene of HIV-2 in the serodiagnosis of HIV-2 infections

We produced recombinant envelope-derived peptides of HIV-2 for use in a diagnostic enzyme-linked immunosorbent assay (ELISA) or Western blot by expressing several restriction enzyme fragments from the env gene of HIV-2 in the bacterial fusion vector pEX-3. On Western blots, 17 out of 18 anti-HIV-2-positive sera available to us reacted strongly with those recombinant peptides which were derived from, or extended into, the transmembrane protein of HIV-2. In contrast, recombinant peptides derived from the external envelope glycoprotein were only weakly recognized by two sera. We observed a cross-reactivity of some human sera containing antibodies to HIV-1 with the HIV-2 peptides derived from the transmembrane protein of HIV-2. In spite of this cross-reactivity, a serological distinction between anti-sera to HIV-1 and HIV-2 can be attempted by simultaneous testing in ELISA on recombinant peptides derived from the transmembrane protein of HIV-1 and HIV-2.     

Bacterially produced HIV-2 env polypeptides specific for distinguishing HIV-2 from HIV-1 infections

Five unique recombinant polypeptides, each encoded by a DNA segment representing a different region of the HIV-2 (NIH-Z strain) env gene, were produced at relatively high levels (greater than or equal to 5%) as cII-fusion products in Escherichia coli. These recombinant polypeptides were characterized serologically by the Western blot assay against a panel of HIV-2 and HIV-1 antibody-positive sera, and with normal human sera (HIV-1 and HIV-2 antibody negative). Only those polypeptides that are encoded by a segment of the env gene from the N-terminal region of the transmembrane protein gp35 (amino acids 537 to 707) were immunoreactive. Three polypeptides (921, 996, and 997), each encoding this immunoreactive region of the HIV-2 (NIH-Z) gp35, reacted strongly and specifically with antibodies in sera from HIV-2-positive individuals, but not with antibodies in sera from HIV-1-positive or HIV-uninfected individuals. These results show that the N-terminal region of the HIV-2 gp35 contains a highly antigenic determinant which is strongly immunogenic in HIV-2-infected individuals. The gp35-encoded recombinant env polypeptides can potentially be used in diagnostic assays to specifically differentiate between HIV-2 and HIV-1 infections.     

Mapping of immunodominant epitopes of the HIV-1 and HIV-2 integrase proteins by recombinant proteins and synthetic peptides.

Different parts of the human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2) integrase proteins were expressed as TrpE fusion proteins in Escherichia coli and used to screen human sera. In the immunoblot, all HIV/integrase-positive human sera tested reacted with the carboxy-terminal third of the integrase protein. Furthermore, they crossreacted with the same part of the heterologous protein. Half (50%) of the HIV-1/integrase-positive sera additionally detected antigenic epitopes in the amino-terminal third of the HIV-1 protein. Two of the recombinant proteins were used to generate polyclonal rabbit sera, which react with type-common epitopes of both integrase proteins. To map the B-cell epitopes of the HIV integrase proteins in more detail, overlapping decapeptides representing the entire integrase proteins of HIV-1 and HIV-2 were synthesized and used in a pin-based oligopeptide ELISA to scan human sera. This method can define three potential immunogenic epitopes of the HIV-1 integrase and one potential epitope of the HIV-2 integrase. The immunodominant epitopes of the HIV-1 integrase, one localized in the amino-terminal (IDKAQDEHEKYHSNWRAM), one in the central (QMAVFIHNFKRKGGIGGY), and one in the carboxy-terminal (AVVIQDNSDIKVVPRRK) part of the protein were synthesized as oligopeptides and used to test a larger panel of human sera in ELISA (156 HIV-1+ sera and 104 HIV-1- sera). The amino- and the carboxy-terminal epitopes were of equivalent reactivity, while the central part of the HIV-1 integrase seems to be less immunogenic. Nearly 90% of the HIV-1/integrase-positive human sera could be detected by a combination of these three peptides.     

Cross-reactivity on western blots in HIV-1 and HIV-2 infections

To examine cross-reactivity of antibodies to heterologous antigens, on HIV-1 and HIV-2 Western blots, we tested sera from 1362 consecutive tuberculosis (TB) patients and 2127 consecutive blood donors. Specimens positive on enzyme-linked immunosorbent assay (ELISA) for HIV-1 or HIV-2 were further characterized by synthetic peptide-based tests, and tested by HIV-1- and HIV-2-specific Western blots. Dual serologic reactivity on synthetic peptide tests was proportionately more frequent in HIV-positive TB patients than in blood donors, and HIV-2 reactivity less frequent. Positive HIV-1 Western blots were seen in 73-83% of specimens specifically characterized as positive for HIV-2 on synthetic peptide tests. Cross-reactivity to HIV-2 Western blots by HIV-1-positive specimens was significantly more frequent in TB patients (35%) than in asymptomatic donors (9%; P less than 0.001). Using recently recommended criteria for HIV-2 Western blot interpretation (presence of two env bands) reduced the overall proportion of HIV-1-positive specimens having a positive HIV-2 Western blot from 27.5 to 16.4%, with [corrected] minimal effect on sensitivity in the diagnosis of HIV-2 reactivity on specimens positive on synthetic peptide tests.     

Epitope location of 13 anti-gag HIV-1 monoclonal antibodies using oligopeptides and their cross reactivity with HIV-2

A series of 15-mer oligopeptides which overlapped by five amino acids (AA) across the p24 of HIV-1SF2 and a similar series across the p18 of HIV-1SF2 were used to identify the locations of 13 anti-gag monoclonal antibodies (MAbs). Three anti-p24 MAbs recognized sequences within the first 50 AA of the amino-terminal. Another anti-p24 recognized a conformational epitope in the centre of the protein and this MAb cross-reacted with two HIV-2 isolates suggesting conservation of this epitope between HIV-1 and HIV-2. One anti-p24 MAb recognized a linear sequence in the carboxy-terminal 100 AA and one p24 antibody was assumed to recognize a truly conformational epitope as it did not react with any of the linear peptides. Four anti-p18 MAbs were located at the carboxy-terminus of p18 with another MAb mapping slightly inwards from the carboxy-terminus and one anti-p18 MAb failed to bind to the p18 peptides. The carboxy-terminal distribution of the p18 MAbs indicated a highly immunogenic nature for this region in mice. None of the anti-p18 MAbs showed cross-reactivity with HIV-2 isolates, confirming the greater sequence variability of p18 over p24.     

The Karpas AIDS Cell Test compared with an enzyme-linked immunosorbent assay for detecting antibody to the human immunodeficiency viruses (HIV-1 and HIV-2)

We have compared the Karpas AIDS Cell Test for antibodies to the human immunodeficiency viruses (HIV) with a commercial enzyme-linked immunosorbent assay (ELISA) (Organon Teknika) by testing serum samples from 324 intravenous drug abusers in Turin. The cell test was found to be more sensitive and as specific as the ELISA with the serum samples from the drug abusers. In Lisbon, 30 samples were tested on slides containing cells infected with HIV-1 and/or HIV-2. All 15 samples, which were positive for HIV-2 alone (in the HIV-2 Elavia test and by the Western blotting technique), were also positive in the Karpas AIDS test. In contrast, only one of the 15 samples (7%) gave a positive reading in the ELISA for HIV-1. Results of 30 samples tested in Turin and Lisbon by the Western blotting technique agreed closely with those obtained with the Karpas AIDS Cell Test. We were also able to show that the entire test can be performed at room temperature and completed within 1 hour. Moreover, the cell test requires minimal skill and simple equipment and is inexpensive. It also includes non-infected cells as a control and the specificity of positive samples may be verified with a bench microscope. Furthermore, this test which detects antibodies to both HIV-1 and HIV-2 allows rapid typing of the infecting strain.     

HIV-1 neutralizing monoclonal antibodies induced by a synthetic peptide.

We have developed a series of murine monoclonal antibodies to a region of the 120 kD envelope glycoprotein (gp120) of human immunodeficiency virus type 1 (HIV-1). This region has previously been implicated as a site for virus neutralization by antisera raised to recombinant proteins and by antibodies made to full-length gp120 purified from virus. The antigen employed was a synthetic peptide containing 15 amino acids, representing amino acid residues 308-322, RIQRGPGRAFVTIGK, of env gp120 (HTLV-IIIB isolate). Five of the monoclonal antibodies raised to this antigen have reactivity with gp120 from divergent strains of HIV-1 in Western blot assays. The two of these five which were tested with live cells infected with the divergent HIV-1 isolates IIIB, MN, and RF were specifically reactive by fluorescence analyses with cells infected with the MN and IIIB isolates. Four of the five monoclonal antibodies blocked the fusion of IIIB-infected cells with uninfected MOLT-4 target cells. The monoclonal antibody most reactive with MN-infected cells by fluorescence, #5025A, blocked the fusion of MN-infected cells with uninfected MOLT-4 cells. Four of the five monoclonal antibodies neutralized the IIIB isolate of HIV-1 in vitro, but none neutralized the MN or RF isolates at the levels of antibody tested (less than or equal to 50 micrograms/ml). Taken together these data indicate that monoclonal antibodies to the immunodominant neutralizing domain of HIV-1 gp120 display different levels of group reactivity depending on the assay system being examined.     

The conserved carboxy terminal region of HIV-1 gp120 is recognized by seronegative HIV-exposed people

OBJECTIVE: To screen HIV-positive, long-term exposed seronegative and low-risk individuals for the presence of antibodies against regions of HIV-1 gp120 that share some degree of homology with HLA. METHODS: Sera were obtained from 63 HIV-1-infected subjects [52 Centers for Disease Control and Prevention (CDC) stage 2 and 11 stages 3/4], 32 HIV-exposed uninfected (HEU) subjects and from 24 low-risk HIV-1 seronegative individuals. They were tested by a peptide-based enzyme-linked immunosorbent assay (ELISA) for reactivity against peptides derived from the HIV-1 gp120 C-terminal region that contain regions of MHC sequence/structural similarity. Ten randomly selected sera from each group were also screened for anti-class I antibodies. RESULTS: Thirty per cent of the long-term HIV-1-exposed seronegative individuals had antibodies against the conserved C-terminal region (C5) of HIV-1 gp120. However, sera from HEU individuals showed no reactivity against other peptides derived from the C2 region of gp120, also an HLA homologous region. Anti-C terminal gp120 antibodies were mainly of IgM subclass, although IgG-specific antibodies were also present. In addition, 70% of HEU individuals had antibodies to HLA class I molecules compared with 15% of HIV-positive patients (restricted to only those HIV-positive patients with anti C-terminal antibodies). CONCLUSION: Our results suggest that antibody responses against the C-terminal region of HIV gp120 and HLA class I may represent markers of apparent natural protection against HIV-1 infection.     

Enhancement of soluble CD4-mediated HIV neutralization and gp 120 binding by CD4 autoantibodies and monoclonal antibodies

We have identified 6 sera containing autoantibodies to CD4 in 174 human immunodeficiency virus-type (HIV-1) positive sera tested in an antigen-capture enzyme-linked immunosorbent assay (ELISA) using sCD4, and none in 34 HIV type 2 sera. These autoantibodies do not bind to cellular CD4, but react with sCD4 to increase its binding in ELISA to monoclonal antibodies and the HIV surface glycoprotein gp120. The effect of CD4 autoantibodies is mimicked by monoclonal antibodies to the third and fourth domains of CD4. The enhanced sCD4 binding to gp120 in ELISA is reflected by a reduction in the concentration of sCD4 required to neutralize HIV-1 and HIV-2 infection in tissue culture when CD4 autoantibodies or the relevant monoclonal antibodies were present.     

Production and characterization of a mouse monoclonal antibody against the Gag p26 protein of human immunodeficiency virus type 2: identification of a new antigenic epitope

One anticapsid (p26) mouse monoclonal antibody was developed after immunization with recombinant p26 Gag protein and was tested for reactivity with different HIV-1 and HIV-2 isolates by ELISA and Western blot analysis. This antibody, named R26.1, reacted with all HIV-2 isolates tested and with recombinant p26 proteins, but no HIV-1 isolates. The epitope of antibody R26.1 was mapped to residues 50-71 in the N-terminal domain of the capsid protein, a highly conserved region in all HIV-2 isolates sequenced to date. This monoclonal antibody may be useful for the detection of HIV-2, and for the discrimination between HIV-1 and HIV-2 infections. Likewise, the identified epitope may be useful for the detection of p26 antibodies in HIV-2-infected individuals.     

Discrimination of HIV-2 infection from HIV-1 infection by western blot and radioimmunoprecipitation analysis

Serum and plasma samples were collected from blood donors who were confirmed positive for antibodies to HIV-1 in the United States, and from blood donors and individuals in West Africa and Portugal who were positive for antibodies to HIV-1, HIV-2, or both. Western blots and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) radioimmunoprecipitation assays (RIPA) utilizing native HIV-1 and HIV-2 proteins were performed on these specimens to determine the ability of these procedures to discriminate between HIV-1 and HIV-2 infections. Extensive serologic cross reactivity between HIV-1 and HIV-2 p24 was found in both populations. Antibody reactivity to the envelope protein gp120 was able to discriminate 20 of 20 (100%) U.S. specimens as HIV-1 infections. In specimens from West Africa and Portugal, Western blot and RIPA were in complete agreement on 33 of 42 samples (78.6%). Among these 33 specimens, 10 were found to be reactive for antibodies to HIV-1 only, 10 were reactive to HIV-2 only, and 13 were considered to be dually reactive, having antibodies reactive with both HIV-1 gp120 and HIV-2 gp120. Nine of the 42 specimens were discordant by Western blot and RIPA classification, being dually reactive by one procedure and reactive with only one viral gp120 by the other technique. Because of the serological cross reactivities between HIV-1 and HIV-2, in certain populations it is difficult to ascertain whether an individual is infected with HIV-1, HIV-2, a new viral type, or whether the individual is infected simultaneously with multiple viruses. More specific tests such as viral isolation or molecular probes may be necessary to distinguish between infections with these viruses in certain populations.     

Discrimination between HIV-1 and HIV-2-seropositive individuals using mouse monoclonal antibodies directed to HIV transmembrane proteins

Mouse monoclonal antibodies directed against the transmembrane proteins of HIV-1 or HIV-2 provided site-directed, unambiguous discrimination between HIV-1 and HIV-2 antibody-positive sera, when employed in immunoassays as competitive probes against serum antibodies. These monoclonal antibodies mapped to epitopes outside of the well-characterized immunodominant regions (IDR) of the transmembrane proteins. The monoclonal competitive immunoassay was a superior method for discrimination compared with immunoprecipitation of metabolically radiolabeled HIV envelope glycoproteins, Western blot against viral envelope glycoproteins, or noncompetitive enzyme immunoassays employing HIV recombinant transmembrane proteins or synthetic IDR peptides as serological targets. The monoclonal competitive assay was not affected by antigenic cross reactivity or nonspecific reactivity exhibited by selected serum samples toward envelope proteins or peptides, respectively. Results of the monoclonal competitive immunoassay were supported by results of a peptide inhibition assay employing free IDR peptides in competition with IDR peptides on a solid support for binding of serum antibody. IDR peptide inhibition clearly demonstrated non-cross-reactive antigenic specificity of sera toward either the HIV-1 IDR or the HIV-2 IDR. The monoclonal competitive assay also identified samples containing antibody to both HIV-1 and HIV-2 transmembrane proteins. Analysis of these samples by IDR peptide inhibition indicated they contained two distinct, non-cross-reactive populations of antibodies, one directed to the HIV-1 IDR and the other directed to the HIV-2 IDR.     

Antigenic domains of the HIV-1 vif protein as recognized by human sera and murine monoclonal antibodies.

To analyze the vif antibody response in individuals infected with the human immunodeficiency virus type 1 (HIV-1) and to determine antigenic epitopes on the vif protein, 104 HIV-1+ sera were screened for reactivity with a recombinant vif protein; 30 (28.8%) of these sera recognized the recombinant vif protein in immunoblot and were employed, together with 17 HIV-1/vif-negative control sera, in an enzyme immunoassay (EIA)-based epitope scanning assay with 183 overlapping decapeptides that covered the complete amino acid sequence of the HIV-1 vif protein (strain BH10). Of the 30 HIV-1/vif+ sera, 87% reacted with decapeptides comprising the two following epitopes: IEWRKKRY (vif amino acids 87-94) or DRWNKPQ (vif amino acids 172-178). The two epitopes were 89% and 100% conserved among different HIV-1 strains and their antigenicity could be confirmed by computer-assisted predictions of vif antigenic determinants. All the sera reactive with recombinant vif protein and with vif peptides originated from patients in CDC stages III or IV. Two murine anti-vif monoclonal antibodies reacted only with the seven C-terminal amino acids of the vif protein (SHTMNGH), which were not recognized by any of the human sera. Our results may be useful for further studies of vif seroreactivity and for the production of anti-vif mono- or polyclonal antibodies using vif peptides.     

Site-directed enzyme-linked immunosorbent assay with a synthetic simian immunodeficiency virus SIVmac peptide identifying antibodies against the HIV-2 transmembrane glycoprotein

A 23-amino-acid-long peptide (AIEKYLEDQAQLNAWGCAFRQVC) representing the transmembranous protein gp32 in SIVmac was used in site-directed enzyme-linked immunosorbent assay (ELISA) for detection of HIV-2-specific antibodies in 567 sera from Bissau, Guinea Bissau. Ninety out of the 567 sera were identified to contain HIV-2 antibodies by whole antigen ELISA and Western blot assays. The peptide ELISA correctly identified 89 out of these 90 seropositives (sensitivity 98.9%). Three sera falsely interpreted to be positive were encountered (specificity 99.4%). The HIV-2 peptide was also used for testing of 93 HIV-1-positive Swedish sera. None of these sera reacted. Site-directed serology employing synthetic peptides should be considered for application as a screening assay.     

Hyperimmune antisera against synthetic peptides representing the glycoprotein of human immunodeficiency virus type 2 can mediate neutralization and antibody-dependent cytotoxic activity.

Twenty-five 13- to 35-amino-acid-long peptides representing regions of human immunodeficiency virus type 2 (HIV-2), strain SBL6669, envelope proteins were evaluated for their immunogenic activity in guinea pigs. The peptides were selected to provide homologous representation of sites in the HIV-1 envelope proteins that were previously documented to have a particular immunogenic importance. A number of the HIV-2 peptides were found to be capable of inducing strain SBL6669 neutralizing and antibody-dependent cellular cytotoxicity (ADCC) antibodies. Two overlapping peptides covering amino acids 311-337 representing the central and C-terminal part of the variable third (V3) region, terminology according to Modrow et al. [Modrow, S., Hahn, B., Shaw, G. M., Gallo, R. C., Wong-Staal, F. & Wolf, H. (1987) J. Virol. 61, 570-578], showed the most pronounced capacity to induce neutralizing antibodies. One of the peptides (amino acids 318-337) also induced antibodies mediating ADCC. Two additional regions in the large glycoprotein, gp125, containing linear sites reacting with neutralizing antibodies were identified (amino acids, 119-137 and 472-509). The transmembrane protein, gp36, of HIV-2 harbored two regions of importance for induction of neutralizing antibodies (amino acids 595-614 and 714-729). ADCC activity was induced by two additional gp125-specific peptides (amino acids 291-311 and 446-461). Thus, except for the single V3-specific site there was no correlation between linear immunogenic sites stimulating neutralizing antibody and ADCC activity. These findings pave the way for development of synthetic vaccines against HIV-2 and possibly also simian immunodeficiency virus infections. The capacity of such a product to induce protective immunity can be evaluated in macaque monkeys.     

Characterization of murine monoclonal antibodies to HIV-1 induced by synthetic peptides

We have used short synthetic peptides, 12 and 13 amino acids in length, conjugated to carrier proteins to develop monoclonal antibodies (MAb) to the envelope glycoprotein of 120 (kD) (gp120) and the 3' open reading frame protein (3-orf) of the human immunodeficiency virus type 1 (HIV-1). The peptides employed were chosen because of their strong hydrophilicity and in the case of the gp120 peptide because it represents a highly conserved hydrophilic region in the envelope protein. The MAb developed displayed appropriate specificities with their respective peptides and reacted with appropriate HIV-1 components (i.e., a 120 kD glycoprotein and a 27 kD protein, respectively) as determined by Western blot analysis. In indirect immunofluorescence assays the MAb strongly stained syncytia present in cultures of HTLV-3B-infected H9 cells. The MAb to the envelope component reacted with the RF isolate of HIV-1, as well as with the 3B isolate in immunofluorescence.