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Synthetic peptides were used to identify continuous antigenic sites on the external envelope glycoprotein gp125 of human immunodeficiency virus (HIV)-2. Initially, seven HIV-2-positive human serum samples were screened with 172 sequential nonapeptides containing a six-amino-acid overlap. This represents the entire gp125 molecule of HIV-2ISY. The antibody reactivity was found to be mainly restricted to 14 regions within gp125. Following these results, 33 longer peptides, 15-24 amino acids in length, were synthesized and tested against a larger number of samples. Eleven antigenic regions were thus identified. Two of these were detected within a region corresponding to the C1 region and four others within a region corresponding to the C2 region of HIV-1. The highest frequency of reactivity (90%) of 31 HIV-2 seropositive human serum samples was elicited by three peptides from a region corresponding to the V3 region of HIV-1. The C-terminal portion of this region was recognized by almost 80% of the samples. Reactive regions corresponding to the V4, V5, and N-terminal portion of V4 were also identified. A mouse monoclonal antibody reacting with gp125 was mapped to the N-terminal region of the molecule and was found to react with the sequence DVWNLFETS. The peptides were used to evaluate the antibody response of monkeys immunized with whole killed HIV-2 or simian immunodeficiency virus (SIV). The monkeys showed a pattern of reactivity similar to HIV-2-infected human serum samples. Postinfection samples from monkeys inoculated with HIV-2 or SIV reacted mainly to peptides from the V3 region. Two peptides were used to detect the seroconversion of two SIV-infected monkeys. Thus, we have demonstrated that human seroreactivity to HIV-2 gp125 occurs at a few distinct linear antigenic sites distributed at similar positions on the molecule as those in HIV-1 gp120.

The present study demonstrates that maternal antibodies to certain epitopes of human immunodeficiency virus 1 (HIV-1) proteins are associated with a defined outcome for at-risk pregnancies of HIV-infected women. An initial retrospective analysis of antibodies to synthetic peptides and recombinant proteins representing env, pol, and gag regions of HIV-1 was carried out. Sera studied were from 33 children who were born to HIV-infected mothers and whose clinical outcome was known at the time of analysis. Sera, collected within the first 6 months of life, of uninfected at-risk children were found to selectively contain maternal antibodies to certain peptides containing epitopes of the HIV envelope glycoprotein gp120. To confirm the predictive role of maternal antibodies to defined HIV-1 epitopes, a prospective analysis was then performed on sera from 21 HIV-seropositive mothers and their infants, whose clinical and immunological status was then followed up for a period of at least 15 months. As expected, antibodies to the same envelope protein peptides were detected almost exclusively in sera from mothers of uninfected children. Our data suggest that antibodies against select epitopes of HIV envelope protein gp120 might play an important role in preventing mother-to-child transmission of HIV-1 infection. Accordingly, site-directed serology might be used to predict the outcome of an at-risk pregnancy of an HIV-infected woman.

To define the amino acids involved in IgG subclass reactivity to two overlapping HIV-1 gp41 (E34/32; amino acid positions 582-613) peptides, sera from 18 HIV-infected individuals were studied. Peptides mimicking E34 but with single amino acid deletions or glycine substitutions were used to define the amino acid residues necessary for antibody binding. Two dominating immunogenic epitopes, containing highly hydrophilic amino acids, were found on the original peptide. Further analysis was undertaken with two corresponding omission sets of dodecapeptides representing halves of the complete E34 plus a terminal cystein peptide. The subclass reactivities usually differed between the patients with regard to the epitopes with which the different IgG subclasses reacted and also to the importance of different amino acids in antibody binding. The 600 glycine and the 601 lysine were involved in the binding of all IgG1, 2 and 4 and most IgG3. The development of E34/32-reactive IgM and IgG subclasses showed different patterns in four patients with primary HIV infections, contradicting the existence of a general pattern for the development of IgG subclasses to this peptide. The findings suggest that different progenitor clones are selected for synthesis of the different subclasses.

Fifteen amino acid peptides, sequentially overlapping by 10 amino acids, were synthesized on the basis of the HTLV-III sequences of the gag and env proteins. They were used as antigens in IgG subclass ELISAs and T-cell stimulation assays. Sera and cells were obtained from 30 asymptomatic, HIV-infected homosexuals. In all subclasses reactivity was found to parts of the gag protein, while IgG1 dominated anti-env peptide responses. It was possible to delineate peptides showing restricted IgG subclass responses that were dominated by either IgG1, 2, 3 or 4. A negative correlation was generally observed between B-cell and T-cell reactivity, but a T-cell and B-cell co-operation was suggested by the response to two IgG1-restricted peptides. The IgG3-dominated epitopes were present in peptides previously known to be amphipathic and capable of T-cell stimulation. The analysis of subclass-restricted responses on the peptide level will assist the understanding of the subclass expression in vivo, since the peptide mapping approximates the delineation of a subclass-restricted response at the level of single epitopes.

Peripheral blood lymphocytes from healthy, HIV sero-negative blood donors have been in vitro immunized using penv9, a recombinant fragment of the envelope of HIV-1. This primary in vitro immunization followed by Epstein-Barr virus (EBV) transformation and somatic cell fusion subsequently gave rise to several specific anti-penv9 monoclonal antibodies (MO28, MO30 and MO43) of mu isotype. The hybridomas have been kept in culture for over 6 months and the antibody productivity for MO30 was measured to 18 micrograms x (24 hr x 10(6) cells)-1. The fine specificity of the antibodies was mapped by a peptide inhibition enzyme immunoassay, using overlapping synthetic pentadeca peptides covering the whole penv9. These human monoclonal antibodies exhibited a similar epitope specificity directed against a non-sequential determinant, including the amino acids 632-646, 677-681 and 687-691. This specificity is very rarely found in immune sera from seropositive patients and presently not reported in human monoclonal antibodies derived from in vivo immunized individuals, indicating that different antibody specificities can be obtained by the in vitro immunization technology. These human monoclonal antibodies did not neutralize HIV. The results presented here demonstrate the feasability of generating human monoclonal antibodies against HIV by primary in vitro immunizations, thereby avoiding the use of lymphocytes derived from infected patients when human monoclonal antibodies for therapeutic purposes are to be produced.

Peripheral blood lymphocytes from healthy, HIV sero-negative blood donors have been in vitro immunized using penv9, a recombinant fragment of the envelope of HIV-1. This primary in vitro immunization followed by Epstein-Barr virus (EBV) transformation and somatic cell fusion subsequently gave rise to several specific anti-penv9 monoclonal antibodies (MO28, MO30 and MO43) of μ isotype. The hybridomas have been kept in culture for over 6 months and the antibody productivity for MO30 was measured to 18 μg × (24 hr × 106 cells)-1. The fine specificity of the antibodies was mapped by a peptide inhibition enzyme immunoassay, using overlapping synthetic pentadeca peptides covering the whole penv9. These human monoclonal antibodies exhibited a similar epitope specificity directed against a non-sequential determinant, including the amino acids 632–646, 677–681 and 687–691. This specificity is very rarely found in immune sera from sero-positive patients and presently not reported in human monoclonal antibodies derived from in vivo immunized individuals, indicating that different antibody specificities can be obtained by the in vitro immunization technology. These human monoclonal antibodies did not neutralize HIV. The results presented here demonstrate the feasability of generating human monoclonal antibodies against HIV by primary in vitro immunizations, thereby avoiding the use of lymphocytes derived from infected patients when human monoclonal antibodies for therapeutic purposes are to be produced. [ABSTRACT FROM AUTHOR]

The article summarizes the work from many laboratories in the identification of antigenic regions from HIV, a variety of methods of identification and interpretation of results means that not all groups agree on the importance of certain epitopes. However, consensus agreement on many regions exist where independent laboratories have confirmed results from others. The knowledge of these regions can be applied in several ways to improve understanding of the natural immune response to HIV, to contemplate manipulation of immune responses in infected individuals, either stimulation as active immunotherapy or suppression of deleterious responses, and in the design of a subunit prophylactic vaccine.