Search

The present study evaluated the combination of antithymocyte globulin (ATG) and cyclosporine A (CsA) in patients with low-risk myelodysplastic syndromes. Twenty patients (17 with refractory anemia and 3 with refractory anemia with excess blasts) received treatment with rabbit-ATG plus CsA. The overall response rate was 30% (6/20); three of the six responders had a complete response. The responses lasted 2-58 months, with two patients still being in complete remission at 42 and 58 months. Short-lasting cytogenetic remissions were achieved in two patients. ATG was poorly tolerated in patients over 70 years of age. Four out of 20 patients progressed to acute myeloid leukemia within a year. We conclude that immunosuppressive treatment may be a therapeutic option for selected patients with myelodysplastic syndrome.

During follow-up after allogeneic stem cell transplantation (SCT), patients frequently lose their immunity to infectious agents such as measles. The aim of this study was to analyze the influence of different factors on measles immunity. In total, 395 patients with a disease-free survival of at least 1 year were included. Measles vaccination was given at 2 years after SCT to children and young adults without chronic GVHD or ongoing immunosuppression. In all, 264 patients had matched sibling donors and 131 either mismatched family or unrelated donors. Totally, 318 patients received bone marrow and 77 peripheral blood stem cells. Overall, 375 patients had undergone myeloablative and 20 nonmyeloablative conditioning. Out of 395 patients, 133 (34%) were seronegative to measles. In multivariate models, younger age or being vaccinated to measles, rather than previous measles disease, before transplantation were risk factors both for becoming seronegative and to have doubtfully protective immunity to measles. Acute GVHD grade II-IV was a risk factor for seronegativity and blood stem cells a risk factor for doubtfully protective immunity. Children and young adults previously immunized to measles have a high risk for becoming vulnerable to a measles infection. Since measles is again circulating in many countries and measles is a serious infection after SCT, vaccination should be considered.

This phase II study determined the efficacy and safety of alemtuzumab, a humanized anti-CD52 monoclonal antibody, delivered subcutaneously as first-line therapy, over a prolonged treatment period of 18 weeks in 41 patients with symptomatic B-cell chronic lymphocytic leukemia (B-CLL). Injections were administered subcutaneously 3 times per week, from week 2 to 3 onward. An overall response rate (OR) of 87% (95% CI, 76%-98%; complete remission [CR], 19%; partial remission [PR], 68%) was achieved in 38 evaluable patients (81% of intent-to-treat population). CLL cells were cleared from blood in 95% patients in a median time of 21 days. CR or nodular PR in the bone marrow was achieved in 66% of the patients and most patients achieved this after 18 weeks of treatment. An 87% OR (29% CR) was achieved in the lymph nodes. The median time to treatment failure has not yet been reached (18+ months; range, 8-44+ months). Transient injection site skin reactions were seen in 90% of patients. Rigor, rash, nausea, dyspnea, and hypotension were rare or absent. Transient grade IV neutropenia developed in 21% of the patients. Infections were rare, but 10% patients developed cytomegalovirus (CMV) reactivation. These patients rapidly responded to intravenous ganciclovir. One patient, allergic to cotrimoxazole prophylaxis, developed Pneumocystis carinii pneumonia. Alemtuzumab is highly effective as first-line treatment in patients with B-CLL. Prolonged treatment is important for maximal bone marrow response. Subcutaneous administration induced very few "first-dose" flulike symptoms and may reduce health care costs in comparison with the intravenous infusions.

Background: The immune response to HIV infection has been extensively studied and the antibody response against the virus has been characterized in detail. It is, however, still unclear which immune function it is most important to stimulate when administering a vaccine against HIV., Objectives: To analyze the functional antibody responses in asymptomatic HIV-1-infected individuals after vaccination with rgp160., Study Design: Forty-nine asymptomatic HIV-1-infected individuals were followed for 9 months and analyzed for changes in functional antibody responses. Forty of them received HIV-1 envelope rgp160 injections and nine did not., Results: Increased levels of antibodies mediating neutralization and cellular cytotoxicity (ADCC) could be seen in subjects who also showed a better CD4 development compared with the patients without increased levels of functional antibodies. Out of nine matched HIV-infected and influenza-immunized controls, none had increased neutralizing activity and only one had an increased ADCC titer. An increased capacity to block soluble CD4 binding to gp120 occurred in 10 immunized patients. Seroreactivity and avidity maturation were detectable to peptides representing consensus HIV-1 envelope regions, indicating an anamnestic response to the patients own virus., Conclusions: The humoral immune response in HIV-1-infected individuals was moderately influenced by repeated gp160 immunizations, while previous studies have shown that HIV-specific T-cell reactivity was strongly increased.

A potential component that may be useful for passive immunotherapy for HIV-1 is human monoclonal antibodies (HumAbs) possessing potent anti-HIV-1 activity that is directed against conserved regions of the envelope glycoprotein. Such antibodies would, in principle, have the ability to neutralize diverse isolates of HIV-1. To develop such reagents, hybridomas were derived by initial Epstein Barr virus transformation of peripheral blood mononuclear cells (PBMCs) from an asymptomatic HIV-1 seropositive donor followed by fusion with heteromyelomas, and secreted anti-HIV-1 antibodies were further characterized. The specificity of one HumAb, designated as clone 3, was determined by enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses that indicated reactivity to the transmembrane envelope glyco-protein gp41. Synthetic pentadecapeptides overlapping by 10 amino acids were utilized for epitope mapping of clone 3; a decapeptide GCSGKLICTT in the transmembrane gp41 was identified as the epitope. Clone 3 bound to SupT1 cells infected with HTLV-IIIB in fluorescent activated cell sorting analysis. In addition, in vitro biological assays demonstrated that clone 3 possessed neutralization reactivity against diverse laboratory isolates as well as an AZT-resistant isolate. Therefore, clone 3 reactivity defines a conserved neutralizable site on the HIV-1 transmembrane glycoprotein. Clone 3 and the conserved immunogenic epitope on gp41 could be useful in passive and active immunotherapy for the acquired immunodeficiency syndrome (AIDS).

Mouse monoclonal antibodies with high human immunodeficiency virus type 1 (HIV-1) neutralizing titers were used for passive immunotherapy of eleven late-state HIV-infected patients. In five patients the serum level of the core protein p24 decreased, while in five cases it remained unchanged. The level of viral RNA in plasma as measured by quantitative polymerase chain reaction (PCR) decreased in four cases, was stable in another four, and increased in three cases. An anti-mouse (HAMA) response developed in eight patients and anti-idiotypic antibodies appeared in six. Immune complexes that formed in patient sera during the treatment were shown to contain mostly envelope glycoprotein gp120 which decreased in nine of the eleven treated patients toward the end of treatment. Antibodies inhibiting gp120 binding to CD4 became detectable or increased in six patients during immunotherapy. Serology of the HIV-1 V3 region was studied for both the HIV-1 IIIB and MN strains with no or very small changes in titer or avidity after treatment. No change in neutralizing titers to strain HTLVIIIB was observed in serum samples collected before and after treatment was terminated. In nine of the eleven patients stimulation of the T lymphocytes to proliferate in vitro when activated by phytohemagglutinin (PHA) was shown to be increased compared to before treatment. Increased T-cell proliferation was also noted with several antigens such as HIV-1 recombinant antigens, cytomegalovirus (CMV), tetanus toxoid (TT), and purified protein derivate of mycobacterium tuberculosis (PPD). These findings indicate a decreased total gp120 content in serum, permitting better T-cell activation.

Strong specific T-cell responses to human immunodeficiency virus type 1 (HIV-1) gp160 were induced by immunization with recombinant gp160 (rgp160). It was given as postinfection vaccination to 40 asymptomatic HIV-1 seropositive patients. The participants received 6 doses of 160 micrograms rgp160 administered intramuscularly at 0, 1, 4, 8, 17, and 26 weeks and were monitored for 1 year. Lymphocyte proliferation was performed by cultivating lymphoid cells in vitro with specific antigens and mitogens. After immunization with gp160, specific T-cell proliferative responses were induced in all 40 patients. One week after the sixth immunization at day 180, a substantially increased response was detected in 98% of the patients, with a mean stimulation index value of 195. Furthermore, proliferative responses were also identified, after immunization, against native gp120 and against a peptide representing the V3 region of gp120. In addition to the HIV-specific T-cell responses, increased reactivity to several other non-HIV antigens, including tetanus toxoid, influenza, measles, and cytomegalovirus, were seen after gp160 vaccination. The responses to CMV and measles were interpreted to represent an improved recall antigen response. Such recall antigen responses were few in matched HIV-infected controls immunized with influenza virus only. All patients initially and repeatedly showed a normal capacity of total T-cell activation, evaluated by the mitogen phytohemagglutinin (PHA). The trend in CD4 counts improved in 30 of 40 patients during the year of follow-up. The frequency of increases of proliferative responses to antigens was associated with a better CD4 trend. Addition of zidovudine for 2 weeks after each immunization had no beneficial effects nor did it prevent induction of immune responses. All patients tolerated the immunizations well, and no systemic adverse effects were noted. This is a phase I trial, and no definitive conclusions regarding clinical efficacy can be reached.

The prognostic and protective role of antibodies mediating cellular cytotoxicity (ADCC) and neutralization was evaluated in sera of HIV-1-infected mothers and their consecutively followed children. The presence and titres of ADCC mediating and/or neutralizing antibodies in maternal sera did not predict HIV-1 infection in their respective children. No significant difference in the sera from the children was seen when comparing the presence of neutralizing antibodies between the uninfected and infected children. Stratification of the infected group according to clinical status revealed differences. Only one of 24 AIDS patients had a high neutralizing titre against IIIB. Four patients had a very low titre and the remaining had no detectable neutralizing antibodies at all. In contrast, 10/17 infected non-AIDS children had neutralizing antibodies. Similarly, no significant difference was seen when comparing the presence of ADCC-mediating antibodies between the uninfected and the infected group of children. However, a significantly higher frequency of ADCC was seen in the seropositive non-AIDS children compared with the AIDS children. This study clearly shows that the presence of antibodies mediating ADCC and neutralization in infected children, 0-2 years old, is associated with a better clinical status and delayed disease progression.

Human MoAbs of IgM class were developed against three regions of the HIV-1 envelope. Uninfected donor lymphocytes were immunized in vitro with recombinant protein pB1. Four out of five antibodies were directed to different parts of the V3 region, which contains a major neutralizing site. Two out of these antibodies were directed to more than one amino acid sequence, indicating reactivity to discontinuous sites. Two of the human MoAbs inhibited viral spread between cells in tissue culture, interpreted as reactivities to conserved amino acid sequences exposed during viral maturation. No MoAb neutralized virus, which may be explained by the relatively low avidity of the antibodies. One MoAb was directed to a region containing amino acids participating in CD4 binding. This technique appears to allow formation of antibodies with fine specificities other than those obtained in infected hosts.

Four major neutralizing regions of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein were identified and characterized with a panel of 80 HIV-1 antibody-positive human sera. Levels of neutralizing antibodies against the HIV-1 strains IIIB, SF2, and RF were compared with reactivity in ELISAs against peptides that correspond to certain regions of the HIV-1 envelope. A correlation between high neutralizing activity and strong seroreactivity against specific peptides suggested that the corresponding regions might be involved in neutralization. This was further substantiated by using peptides to inhibit neutralization by a panel of 10 HIV-1 antibody-positive sera. The positions of three neutralizing sites, defined earlier mostly by antisera from animals, were confirmed in the present study. Human sera thus recognize the strain-specific third variable region of gp120 (amino acids 304-318), the C-terminal end of gp120 (amino acids 489-508), and the conserved region in the intracellular part of gp41 (amino acids 732-746). It is likely that these different regions mediate help rather than self-sufficient neutralization. Furthermore, a human neutralizing region was detected in a conserved part of gp41 (amino acids 647-671). Accordingly, neutralizing antibodies directed to this region were found to be cross-reactive between HIV-1 strains. Peptides corresponding to these four regions were able to inhibit neutralization mediated by serum from HIV-1 antibody-positive individuals. These results indicate that this conserved B-cell epitope of the HIV-1 envelope elicits a virus-neutralizing antibody response during natural infection in humans and may therefore be considered for inclusion in a vaccine against HIV-1.

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 importance of the dependence on single amino acids in the V3 region of HIV-1 gp120 was evaluated for virus neutralization and antibody-dependent cellular cytotoxicity (ADCC). Synthetic overlapping 15-mer peptides and a set of omission peptides covering amino acids 301-317 were used. Sera from 29 HIV-1-infected individuals at different stages of disease were tested for neutralization, ADCC and specific IgG reactivity. Six HIV-1 neutralizing monoclonal antibodies (mAb) acted as controls. All mAb reacted with a region (amino acids 304-318) of gp120, previously shown to induce neutralizing antibodies. The amino acids essential for reactivity were identified to be within the sequence GPGR (amino acids 312-315). The importance of this region for occurrence of neutralizing antibodies in infected humans was investigated using the same set of peptides. Out of 29 individuals, 21 were found to have neutralizing antibodies in titres between 100 and 1000. Among the neutralization-positive sera, 17/21 (81%) reacted with amino acids 304-318, compared with only one of eight sera (13%) negative in neutralization. When any of the four amino acids G, P, G or R were deleted, the seroreactivity decreased considerably. The conserved sequence GPGR was therefore considered to be the most important for neutralization in this region in human sera as well. Thus, the conserved sequence GPGR in the V3 region of gp120 is critical for virus neutralization by human HIV-1-specific antibodies.

A panel of highly purified synthetic oligopeptides representing defined parts of the gag and env proteins of HIV-1 and HIV-2 were used as antigens in ELISA for serodiagnosis of HIV-1 and HIV-2 infection. The analysis included sera from 321 HIV-infected patients and 201 healthy controls from the Ivory Coast, where the prevalence is high for both HIV-1 and HIV-2, and sera from European HIV-1-infected individuals. All sera from HIV-1-infected individuals reacted with a 20 amino acid (a.a.) peptide JB-4c (a.a. 594-613) derived from a highly immunogenic conserved region of the external part of gp41. An equally good response was seen in the HIV-2-infected individuals to a 20 a.a. peptide, JB-16c, from the corresponding part of HIV-2 gp36. Both HIV-1- and HIV-2-seropositive individuals responded well to a peptide, JB-8pc (a.a. 427-448), representing the C-terminal end of the putative CD4-binding site of gp120 of HIV-1. The frequency of reactivity to three selected HIV-1 gag peptides derived from p17 and p15 was 60-70% in both HIV-1 and HIV-2-positive sera. To distinguish between HIV-1 and HIV-2 infection, the sera were titrated against the peptides. Although there was a high degree of cross-reactivity at lower serum dilutions, it was possible to discriminate the infections at higher dilutions to the HIV-1 and HIV-2 gp41/gp36 peptides JB-4c and JB-16c. Analysis of serum reactivity to several selected peptides thus allowed the identification of HIV infection, and the discrimination between HIV-1 and HIV-2.(ABSTRACT TRUNCATED AT 250 WORDS)

Five mouse monoclonal antibodies were raised against a recombinant protein comprising the complete sequence of gag24 protein from HTLV-IIIB. All monoclonal antibodies recognized the native protein in enzyme-linked immunosorbant assay (ELISA) and Western blots. All monoclonal antibodies also cross-reacted with an human immunodeficiency virus type 2 (HIV-2) strain in western blots, whereas only one antibody detected HIV-2 p25 in ELISA. By using synthetic peptides, cross-reacting epitopes were mapped and three regions were defined. The conserved immunogenic sites were located in the carboxyterminal region of the protein. Inhibition experiments with human sera showed that this region is also immunogenic in humans.

Amino acid sequences inducing neutralizing antibodies to HIV-1 were sought. Murine monoclonal antibodies (MAbs) were characterized by their reactivity with the envelope precursor gp160 or the Escherichia coli recombinant DNA products pB1 and pE3 representing the carboxy- and amino-terminal halves of mature envelope gp120. Fine mapping of the MAb determinants was performed using defined 15-mer synthetic peptides spanning the entire envelope gp120 region of HIV-1. One group of MAbs recognizes epitopes (amino acids 304-323) occurring in a small region with variable and conserved amino acid sequences of gp120. These MAbs mediate neutralization of the HIV-1 strain HTLV-IIIB (HIV-1IIIB) which was used for immunization. Nine out of 11 primary HIV-1 isolates were neutralized well or moderately well. In addition, prominent serological reactivity was noted with peptide sequences of strains of various European or American origins, but not with two HIV-1 strains of African origin. The cross-reactivity contrasts with previously described type-specific reactions to other sequences of this region. The reactivity to the short conserved site GPGR with its flanking amino acids may explain the broad sequence cross-reactivity seen with our neutralizing MAbs. Two other MAbs recognize conserved epitopes (amino acids 79-103) situated in the amino-terminal region of gp120. These MAbs did not neutralize HIV-1IIIB.

We have obtained 15 HIV-2 isolates from the peripheral blood mononuclear cells (PBMCs) of 24 HIV-2-infected west African people. The frequency of virus isolation correlated with the severity of HIV-2 infection; only three isolates were obtained from 11 asymptomatic individuals, whereas virus was isolated from nearly all (12 of 13) individuals with symptoms. The HIV-2 isolates showed distinct replicative and cytopathic characteristics and, similarly to HIV-1 isolates, could be divided into two major groups: rapid/high and slow/low. Rapid/high isolates, i.e. isolates with the ability to replicate in tumour cell lines, were obtained from individuals with symptomatic HIV-2 infection and CD4+ lymphocyte counts less than 360/microliters blood; these isolates induced syncytia in PBMC cultures. HIV-2 isolates unable to replicate continuously in tumour cell lines (slow/low isolates) induced small syncytia, cell death, or no cytopathic effect at all. All HIV-2 isolates obtained from asymptomatic individuals showed a slow/low replication pattern.

In children born to human immunodeficiency virus (HIV)-infected mothers, factors that determine disease outcome and progression are unclear. Also, early diagnosis is hampered by maternally transferred antibodies. Children aged 0-24 months were retrospectively divided into two groups based on HIV seroreactivity or nonreactivity at age 15 months and analyzed for the presence of antibodies that mediate cellular cytotoxicity (ADCC) and virus neutralization. No difference was seen in the presence of these functional antibodies between groups. The persistently seropositive group was further divided into non-AIDS and AIDS groups according to clinical status at serum collection. The ADCC antibody frequencies were much higher (70%) in the non-AIDS group than in the AIDS group (30%). Of the non-AIDS children, 63% had neutralizing antibodies; no children with AIDS had these antibodies. HIV-specific ADCC and neutralizing antibodies do not seem to protect against transmission of HIV from mother to child but are significantly correlated with a better clinical stage of childhood HIV infection.

Monoclonal antibodies (MAbs) were raised against human immunodeficiency virus type 1 gp120. One MAb, P4/D10, was found to mediate highly efficient antibody-dependent cellular cytotoxicity and virus neutralization. The reactivity was located to a major neutralizing region (amino acids 304 to 323) on gp120. Five other MAbs with a similar epitopic reactivity did not show any antibody-dependent cellulan cytotoxicity activity but had a virus-neutralizing capacity.

Phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, suppresses natural, lectin and antibody-dependent killing by normal human lymphocytes in short-term radioisotope release assays. Fifty percent inhibition of killing of lymphoid target cells was seen at approximately 5 ng/ml TPA and inhibition was further potentiated by the presence of monocytic cells. In contrast, TPA increased killing of K-562 erythroleukaemic cells by non-adherent NK cells with optimal activity around 1 ng/ml. Two anti-estrogenic drugs, tamoxifen and clomiphene, known to inhibit protein kinase C, gave near to complete inhibition of NK killing at concentrations 12 microM and 30 microM, respectively. Retinal, another protein kinase C inhibitor, inhibited both antibody-dependent killing and lectin-dependent killing. An influx of 45Ca2+ into the effector population was found during effector-target cell conjugation and this flux was suppressed at TPA concentrations similar to those that suppressed killing. The results suggest that killing depends on a co-ordinated activation of protein kinase C together with a channel-dependent calcium influx. TPA may suppress killing by a negative feedback effect of protein kinase C on the hydrolysis of inositol phospholipids, as demonstrated in many other systems, or through the down-regulation of cell surface receptors required for triggering of lysis.

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.

Antibodies mediating HIV-1-specific cellular cytotoxicity (ADCC) and virus neutralizing activity were detected in the cerebrospinal fluid (CSF) and, as previously reported, in sera of subjects with varying severity of HIV-1 infection, including patients with or without neurologic or psychiatric symptoms. ADCC-mediating antibodies against target cells infected with the HTLV-IIIB strain of HIV-1 were less frequently present in CSF than in sera, 32 and 60%, respectively. The frequency of ADCC-positive CSF was comparable for the different clinical stages of the disease and was apparently not influenced by the presence or absence of neurologic/psychiatric symptoms. Virus-neutralizing activity was tested against HTLV-IIIB and one CSF-derived viral isolate. Serum antibodies neutralized HTLV-IIIB more frequently than the CSF isolate, 53 and 35%, respectively. In contrast, only three (7%) of the CSF specimens contained neutralizing activity to the CSF-derived isolate and none to HTLV-IIIB. Despite an intact blood-brain barrier in many subjects, the serum/CSF ratios of ADCC or neutralizing antibodies were lower than normally expected. This indicates that both ADCC-mediating and virus neutralizing antibodies may be intrathecally synthesized. Whether these antibodies are protective against or contribute to the histopathologic changes in the brain is not known at present.

We analysed HIV-specific immunoglobulin G (IgG) responses to gag and env peptides in infants born to HIV-positive mothers. Questions of interest were whether there are early specific markers for prognosis, and whether any specific IgG is related to the prevention of vertical transmission of infection. Fifty-three children, 0-24 months old and born to HIV-1-infected mothers, were retrospectively divided into two groups based on HIV seroreactivity or non-reactivity at 15 months of age. Their sera were used to find reactivities important in diagnosis and/or prediction of the putative HIV disease. Three important findings emerged. First, a low IgG titer against the very immunodominant penv9 in newborns was found to be associated with rapid progression to AIDS. This difference was clearly reflected in the reactivity to a small peptide representing amino acid (aa) 598-606. The second interesting finding was the putative hypervariable loop on gp120 (especially aa 324-338), reactivity to which was found only in the uninfected group, and was seen in six out of 19 uninfected children under 6 months of age. This specific response was not caused by a generally high total anti-HIV reactivity, and may indicate a role of protective antibodies against vertical transmission. The response to this region in the infected group, on the other hand, was directed to the amino terminal half of the putative loop, in particular peptide 53, aa 304-318. Finally, response to a part of the amino terminal end of P17 was seen in seven out of eight infected children over 6 months of age.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

There is an association between the clinical stage of HIV-1 infection and the presence of antibodies against viral gag proteins (p17 and p24). The IgG subclass (G1 and G3) pattern against these antigens was analysed in stable patients and HIV patients progressing to AIDS. Antibodies were analysed with whole viral or peptide ELISA (using sequentially overlapping peptides) and Western Blots. IgG1 was found to be the dominant anti-HIV-1 IgG subclass and IgG1 antibodies declined in progressing patients against all HIV antigens evaluated in Western blot, including p17, p24, p31, gp41, p64, gp120 and gp160. In contrast IgG3 antibodies, which were found to be predominantly directed against gag proteins, and which could be detected in almost all patients, remained in the circulation during disease progression. By peptide assays distinct immunogenic regions were found in p17 in contrast to more evenly distributed epitopes in p24. A decreased divergence of antibody reactivity to both p17 and p24 peptides in the group of patients who developed AIDS was seen. No reaction to any single gag epitope related to disease progression. The difference between IgG1 and IgG3 anti-gag antibodies in relation to clearance during disease progression may depend on different properties of immune complexes formed by these two IgG subclasses.

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.

The anti-HIV IgG subclass response was analysed in sera from different clinical stages and related to virus specific antibody-dependent cellular cytotoxicity (ADCC). IgG1 was found to be the dominant subclass, present in all sera and with similar mean titres at different stages. The number of anti-HIV IgG3 positive sera, measured on whole viral lysate antigen plates, decreased during disease progression from 38% in symptom-free to 7% in AIDS patients. IgG2 and IgG4 subclasses were less prevalent although a slight increase of IgG4 frequency was found in AIDS patients. High IgG1 titres correlated with a positive ADCC reaction but there was no correlation between anti-HIV IgG1 and ADCC titres. Some sera which contained HIV IgG1 as the only subclass were able to mediate an ADCC reaction. In addition, when anti-HIV IgG3 was isolated, by protein A chromatography, no ADCC killing was induced by these antibodies. It is concluded that IgG1 is the major ADCC-active IgG subclass in HIV infected individuals. The lack of correlation between IgG1 and ADCC titres may be explained by a relatively small fraction of IgG1 antibodies mediating ADCC.