Combining human antisera to human leukocyte antigens, HIVgp120 and 70 kDa heat shock protein results in broadly neutralizing activity to HIV-1
Babaahmady, Kaboutar; Bergmeier, Lesley A; Lehner, Thomas
From the Kings College London, Mucosal Immunology Unit at Guy's Hospital, London SE1 9RT, UK.
Received 7 January, 2008
Revised 14 April, 2008
Accepted 14 April, 2008
Correspondence to Dr Thomas Lehner, Kings College London, Mucosal Immunology Unit at Guy's Hospital, Guy's Tower Floor 28, St Thomas' Street, London SE1 9RT, UK. Tel: +44 20 7188 3072; fax: +44 20 7188 4375; e-mail: email@example.com
Objective: To elicit broadly neutralizing antibody activity by combining polyclonal human serum IgG antibodies with HIVgp120, human leukocyte antigen (HLA) class I or class II and 70 kDa heat shock protein.
Design: In addition to HIV antigens, HIV-1 virions express HLA class I, HLA class II and 70 kDa heat shock protein molecules, which have quantitative and functional significance. The complementary effect of combining human polyclonal IgG antibodies with these antigens may result in effective broad spectrum neutralizing activity.
Methods: Polyclonal human sera with IgG antibodies and monoclonal antibody to HLA class I or class II, HIVgp120 and 70 kDa heat shock protein were selected and used in single, double or triple combinations. Dose-dependent inhibition studies of HIV-1 clades A, B, C and D were carried out using human CD4 T cells treated with the combinations of human sera and with monoclonal antibodies for clade B. The results are presented as half maximal (IC50) inhibitory concentration and maximum inhibition by these sera.
Results: The half maximal (IC50) inhibitory concentration of clade B HIV-1 infection with single or a combination of two antisera was higher than those with three antisera, which also showed maximum inhibition of HIV-1. Further investigations of human sera with HIV-1 clades C and D also showed lower half maximal (IC50) inhibitory concentrations and higher maximum inhibition with combinations of the three antisera, but this was not seen with clade A.
Conclusion: A novel vaccination strategy eliciting broadly neutralizing antibody activity to the CCR5-using HIV-1 clades B, C and D has been demonstrated by the trimolecular complex of human antisera with HLA class II or class I, HIVgp120 and 70 kDa heat shock protein.
HIV-1 virions bud from the cell membrane and carry with them a non-random selection of host proteins , of which human leukocyte antigen (HLA) class I and II  and 70 kDa heat shock protein (HSP70)  are of special interest. Surprisingly, more HLA molecules are found in the viral envelope than HIVgp120 molecules and the amount of HSP70 is similar to that of viral Pol protein. Targeting the cognate HIV-1 antigens is the classical way of preventing infection, but this approach has encountered serious problems in eliciting effective cytotoxic lymphocytes (CTL) and neutralizing antibodies. Viral escape from CTL and neutralizing antibodies is frequently found and HIV-1 mutation may occur in the acute  and chronic phase . An alternative strategy, which has yet not been explored, is to target HLA and HSP70 host molecules, in addition to HIVgp120 expressed by the virions.
Remarkably, xenoimmunization, arguably one of the most effective and reproducible preventive vaccine strategies against SIV infection, established over 15 years ago, has received limited attention. Although xenoimmunization, unlike alloimmunization, has not been pursued in humans, it offers a model to study the mechanism of protection and to identify correlates of protection. Xenoimmunization in macaques with SIV grown in human CD4 T cell lines induced complete protection (sterilizing immunity) in 85–100% macaques challenged with SIVmac (reviewed in ). However, immunization only with the human cells in which SIV was grown also elicited significant protection from SIV infection, though the degree of protection by human cells alone was lower than that achieved by combination with SIV . Experiments in macaques by immunization with human HLA-II antigens [8,9] or HLA-I purified from a human B lymphoblastoid cell line  induced protective immunity when challenged with SIV grown in human CD4 T cells.
Protective immunity induced by xenoimmunization could be transferred passively with antibodies to HLA-II to naive macaques challenged with SIV [8,9]. Alloimmunization with simian peripheral blood mononuclear cells (PBMC) also prevented SIV infection  but not when a Mamu A1 B cell line was used . Allostimulation of human cells in vitro , women in vivo [13,14] and during unprotected sexual intercourse  demonstrated ex-vivo protection against HIV-1. Limited attention has been paid to HSP70, but both mRNA and expression of HSP70 are upregulated in CD4 T cells infected with HIV-1 . Indeed, HSP70 is found in the virion membrane of HIV-1 and functions as a chaperone during intracellular transport .
In view of the consistent sterilizing immunity induced by vaccination with SIV grown in human CD4 T cell lines, we explored the hypothesis that antibodies to HIVgp120 and two major host antigens, all of which are expressed in HIV-1 virions, may have a potentiating effect in inhibiting HIV transmission. The rationale for this approach was that effective broad-spectrum inhibitory antibody titre has so far been difficult to attain by immunization with a single antigen. Initially, polyclonal human sera with IgG antibodies to HLA-I, HLA-II, human HSP70 and HIVgp120 were used in single, double or triple combinations with the objective of inhibiting clade B HIV-1 replication. The sera showed complementary inhibition of HIV-1 infectivity of activated human CD4 T cells when a combination of HLA-II or HLA-I, HIVgp120 and HSP70 was used and confirmed with the corresponding mouse monoclonal antibodies (mAb). This strategy was then pursued with clade C prevalent in China, southern Africa and India, and clades A and D prevalent in central and eastern Africa. These subtypes of HIV-1 showed a range of inhibition between 73 and 100% with the triple combinations of human IgG antibodies.
Materials and methods
Polyclonal and monoclonal antibodies
The Ig class and source of human serum antibodies to HIV-1 gp120, HLA-I, HLA-II, HSP70 and the control AB serum are shown in Table 1. Sera of each of the following three groups were collected (Table 1): multiparous women (n = 5) with antibodies to HLA-I and HLA-II, donated by the UK Transplant Support Services Authority (Bristol, UK). Screening 20 sera from normal participants, we selected two with antibody titres to human HSP70 of 1: 200. Two sera from HIV-positive men were selected from a joint AmFAR study by Dr P. Anton (UCLA AIDS Institute, Los Angeles, USA); these yielded HIVgp120 antibody titres of 1: 800 and 1: 600. The Ig class and source of mAb to HIV-1 gp120, HLA-I, HLA-II, human and microbial HSP70, and the isotype controls of the IgG1 and IgG2a class are shown in Table 2.
The mAb to HIV-1 gp120 (Abcam Ltd, Cambridge, UK) was an uncharacterized commercial mAb. Preliminary neutralization inhibition studies (×3) were carried out with V1, V2 and V3 peptides, which showed that mAb recognizes the V3 loop (data not presented).
IgG separation was carried out with all human sera by diluting 1 ml serum in 10 ml phosphate buffer of pH 7.0. The sera were then filtered and applied to a 1 ml column of Protein G-Sepharose Fast Flow (GE Healthcare, Amersham, UK). Unbound protein was removed by washing with 20 mmol/l phosphate buffer. The IgG fraction was eluted with 200 mmol/l glycine/HCl pH 2.8 and dialysed against phosphate-buffered saline (PBS). Protein concentrations were determined and 1 mg/ml IgG was filter sterilized through 0.2 μm syringe filters (Amicon, Peterborough, UK).
Culture of HIV-1 clades A, B, C, D and group O
Clades A (92/RW/020 and 92/RW/024), B (HIV-1 BaL), C (93In101 and MW/93/960) and D (94/UG/108 and 94/UG/114) R5 HIV-1 (Table 3) were obtained from the National Institute of Health (NIH; AIDS Research and Reference Reagent Program, Rockville, Maryland, USA) or National Institute for Biological Standards and Control (NIBSC; Potters Bar, UK), LAI and group O (clinical isolate) ×4 strains of HIV-1 were obtained from NIBSC or donated by Dr R. Shattock.
The initial systematic analysis was pursued with clade B (BaL), followed by the other clades when it became evident that a combination of three polyclonal human antibodies elicit greater neutralization of BaL than single or double antibodies. The HIV-1 strains were cell-free supernatants from cultured PBMC and expanded by phytohemagglutinin (PHA) according to standard methods from the NIH database. Culture supernatants were collected and tested for p24 antigen by ELISA using the Retrotek kit (ZeptoMetrix Corporation, Buffalo, New York, USA). Culture supernatants of all strains with high titre were tested on PHA-activated PBMC (3 days) and p24 antigen was assayed at day 7 to calculate the half maximal tissue culture inhibitory concentration (TCIC50) of these stocks of HIV-1 strains.
Dose-dependent inhibition studies
Aliquots of 2 × 105 CD4+ T cells of the PM1 cell line derived from a CD4+ T cell clone and donated by Dr R. Shattock were treated with 25 μl of IgG antibodies to HLA-I, HLA-II, gp120, HSP70 and an isotype control at concentrations of 3.1, 6.25, 12.5, 25 and 50 μg/ml in duplicates for each concentration, as well as without any antibodies . The same dose-dependent concentration of each antibody was used with two or three combined antibodies and control AB serum. Single antibody comparisons were performed in all instances in parallel with two or three combined antibodies. The cells were then treated with the HIV-1 BaL or LAI [10 half maximal tissue culture infective dose (TCID50)] for 2 h, washed three times with medium and cultured in triplicate at 1 × 105 cells per well in 96-well culture plates. Every 2 days, 100 μl of the culture supernatant was replaced with 100 μl of medium, supplemented with appropriate antibody. On day 7, the culture supernatants were used to determine reverse transcriptase activity by the Quan-T-RT assay system (Amersham Life Science, Little Chalfont, UK). The results are presented for the dose-dependent inhibitions as the mean percentage (±SEM) of reverse transcriptase activity (Fig. 1). In addition, the half maximal (50%) inhibitory concentration (IC50) and maximum inhibition achieved by the antibodies are presented in Table 4.
Inhibition assay of HIV-1 primary isolates were then pursued with clades A, B, C and D (R5 strains) and LAI and group O (clinical isolate) of the ×4 strains with single antibodies and combinations of antibodies. Human PBMC were prepared from normal blood on Ficoll-Hypaque gradients. PBMC were activated with 10 μg/ml PHA (Sigma, St Louis, Missouri, USA) and 20 IU interleukin-2 (IL-2) (Schiaparelli Biosystems BV, Woerden, Netherlands) in culture medium of Roswell Park Memorial Institute (RPMI) medium with 10% foetal calf serum (Biosera), penicillin 100 U/ml, streptomycin 100 μg/ml and L-glutamine 2 mmol/l (Sigma) for 3 days and then washed with medium and cultured in RPMI with 20 IU IL-2 overnight. Aliquots (100 μl) of 6 × 105 PBMC were infected with 10 TCIC50 of HIV-1 for 2 h. The cells were washed three times with medium and cultured in triplicate at 2 × 105 cells/well in 200 μl medium and treated with the five concentrations of antibodies in 96-well culture plates. Every 3 days, 100 μl of culture supernatant was replaced with 100 μl of medium, supplemented with antibodies. On day 9, p24 antigen assay was carried out by ELISA according to the manufacturer's instructions (ZeptoMetrix Corporation).
The results are expressed as mean (±SEM) when three or more experiments were carried out. IC50 was calculated by plotting the percentage inhibition against concentrations of antibodies and selecting the concentration of antibodies at 50% inhibition of HIV-1 infectivity. The difference in the IC50 between the triple and single antibodies was evaluated by the Student's t-test for two means.
Inhibition of HIV-1 clade B infectivity of CD4 T cells with polyclonal human IgG antibodies
The aim was to find out whether human HLA-I and HLA-II antisera from multiparous women, HSP70 antisera from normal participants and HIV-1 gp120 antibodies induced in HIV-1-infected persons might yield complementary neutralizing antibodies. Dose-dependent inhibition of HIV-1 with single antisera showed a mean (±SEM) IC50 of 8.0 (±2.8) to 19.0 (±8.4) (Fig. 1a; Table 4) and those with double antisera showed 6.5 (±2.8) to 18.3 (±6.5) μg/ml (Fig. 1b; Table 4). Furthermore, maximum inhibition values with single or double antisera were up to 90%. In contrast, IC50 of the three antisera with HLA-II + HIVgp120 + HSP70 antibodies was 3.8 (±0.6) μg/ml, with a maximum inhibition of 96.7 (±0.9)%, and that of HLA-I + HIVgp120 + HSP70 was 4.7 (±0.4) μg/ml, with maximum inhibition of 92.8 (±3.4)% (Fig. 1c; Table 4). The triple combination of HLA-II + HIVgp120 + HSP70 IC50 (3.8 ± 0.6) was significantly lower (t = 9.80, P = 0.010) than HLA-II alone (IC50 18.2 ± 1.3) or the combined analysis of each of the three corresponding antibodies (IC50 15.1 ± 3.1; t = 3.52, P = 0.008). HLA-I + HIVgp120 + HSP70 also showed a significantly lower IC50 (4.7 ± 0.4) than the combined analysis of the corresponding three antibodies (IC50 12.8 ± 3.3; t = 2.71, P = 0.027).
To exclude the possibility that the inhibitory effect of the combination of three HLA-II or HLA-I antisera was not confined to a specific allele, we examined combinations of four other available human alloantisera (Table 2). These showed IC50 of 3.5–9.0 μg/ml and maximum inhibition of 90.5–97.3% with the antisera to HIVgp120 and HSP70. Thus, although some differences in neutralization of HIV-1 were observed between different human HLA-II or HLA-I antisera, in principle, the combinations of three selected antisera showed higher neutralization of HIV-1 than the constituent single or double antisera. The corresponding antisera also failed to show inhibition with the ×4 strains of HIV-1 (LAI and group O clinical isolate; Fig. 1c).
Inhibition of HIV-1 clade B infectivity of CD4 T cells with monoclonal antibody
To determine the relative magnitude of inhibition, a broadly reactive mAb (b12) was used in parallel with the triple combination of HLA-II + HIVgp120 + HSP70 antisera. This showed that the IC50 of the IgG mAb b12 was 1.75 μg/ml compared with that of 2.25 μg/ml with the HLA-II + HIVgp120 + HSP70 antisera. The mAb was slightly more effective than the polyclonal serum antibodies, which inevitably contain other antibodies. However, to exclude other serum inhibitory factors and unrelated antibody activities, we pursued inhibition studies with the corresponding mAb. Dose-dependent inhibitions of CD4 T cells were carried out with single mAbs and the results were expressed as mean (±SEM) of three to five assays (Fig. 2a–c). The IC50 of single mAb ranged between 11.0 (±6.8) and 36.0 (±7.3), two mAbs gave a range of 7.2 (±2.8) to 30.0 (9.3), and three mAbs showed a range between 3.5 (±1.3) and 7.0 (±2.8) (Table 4). The isotypes IgG1 and IgG2 controls or the combined mAbs failed to significantly inhibit HIV-1 replication. As with the IC50, the triple mAb + HLA-II + HIVgp120 + HSP70 yielded the highest maximum inhibition (99.0 ± 0.5%), followed by HLA-I + HIVgp120 + HSP70 of 83.7 (±9.4%) (Fig. 2c; Table 4).
Thus, targeting the three major molecules of HIV-1 virions resulted in the lowest IC50 and highest inhibition of HIV-1 by the combined IgG mAbs to HLA-II + HIVgp120 + HSP70, which is consistent with the human polyclonal IgG antibodies. Statistical analysis of the difference in IC50 between HLA-II + HIVgp120 + HSP70 (3.5 ± 1.3) and the combined three mAbs (19.7 ± 5.1) was significant (t = 3.07, P = 0.015). Similarly, the difference between HLA-I + HIVgp120 + HSP70 (7.0 ± 2.7) and the three combined mAbs (24.1 ± 5.5) was significant (t = 3.66, P = 0.011). Furthermore, 99.0 (±0.5)% inhibition of HIV-1 replication was attained only by targeting the HLA-II + HIVgp120 + HSP70 molecules expressed by HIV-1 particles.
Inhibition of HIV-1 clades A, C and D
The dose-dependent inhibition studies were then pursued with clades A, C and D in order to find out whether broadly neutralizing antibody function can result by combining the three types of antibodies (Fig. 3). The IC50 of HLA-II + HIVgp120 + HSP70 antibodies was 4.5, 2.5 and 6.1 μg/ml with clades A, C and D, respectively, but it rose to 32.0 and 20.5 μg/ml, respectively, with clade A (92/RW/024) and clade C (93/IN/101) (Fig. 3; Table 5). Similarly, the IC50 of HLA-I + HIVgp120 + HSP70 antibodies was 7.0 μg/ml with clade C and 5.0 μg/ml with clade D but rose to 27.0 μg/ml with another clade C and 27 μg/ml with clade A. It is of some interest that the maximum inhibition elicited by the triple antibodies of clades A, B, C and D was significantly correlated with the IC50 (r = 0.591, P < 0.05), and this was not found with single antibodies (r = 0.441, P > 0.05, data not presented). Although the IC50 and maximum inhibition with either HLA-I or HLA-II combined with HIVgp120 and HSP70 antibodies were greater than those of single antibodies to HIVgp120 with clades C and D, this was not seen with clade A and one of the clade C strains (93/IN/101).
A number of human mAbs to HIVgp120 and transmembrane gp41 (2G12, b12, 2F5 and 4E10) show broad spectrum neutralizing properties [18–21,30–34]. They have been used successfully in passive immunization in macaques, and the complementary effect of two or three mAbs on HIV/SIV chimeric viruses was more effective than single mAb in preventing vaginal [22–25] or neonatal transmission [26–29]. Whether active preventive immunization will elicit high titres of such neutralizing antibodies is uncertain , and some may elicit cardiolipin autoreactivity .
An alternative strategy presented in this study addresses four molecules found in HIV-1 virions, which are unlikely to be conformationally dependent or hidden from the immune system. Antibodies to HLA-I or HLA-II are commonly found in multiparous women resulting from paternal foetal semiallogeneic antigens . Antibodies to HSP70 have been described in normal human participants , probably as a result of dendritic cells taking up HSP70 from T and B cells, especially under conditions of stress , but they have not been tested for HIV-1 inhibition. Antibodies to HIVgp120 envelope can be readily elicited, but usually they do not show broadly neutralizing antibodies. However, a few rare sera have been described from HIV-1-infected individuals who develop broadly neutralizing activity [40–46]. The data presented in this study raise the possibility that rare broadly neutralizing antibodies might be accounted for by a combination of HIVgp120, HLA and HSP70 antibodies.
As the antibodies target HLA-I, HLA-II, HIVgp120 and HSP70 molecules, each of these may affect HIV-1 entry and replication. HIVgp120 antibodies can neutralize HIV-1 and passive transfer of HLA-II antibodies protected macaques challenged with SIV [8,9]. HLA-II molecules promote assembly and budding of HIV-1 at the plasma membrane, and antibodies to these HLA-II molecules may affect HIV-1 replication . Immunization with HLA-I can also protect macaques from SIV infection . HSP70 plays an essential role in virion assembly and uncoating , HIV-1 preintegration complex formation , cell cycle arrest and apoptosis induced by HIV-1 viral protein R (VPR) . Furthermore, ATPase activity of HSP70 is required to maintain HIV-1 virion integrity .
In this study, we show systematically an increase in dose-dependent inhibition of clade B HIV-1 by single, double or triple combinations of antibodies to HLA-I or HLA-II with HSP70 and HIV-1 gp120. The lowest IC50 with clade B HIV-1 of 3.8 (±0.6) μg/ml and maximum inhibition of 96.7 (±0.9)% resulted to a greater extent from a combination of the three antibodies with HLA-II + HIVgp120 + HSP70 and to a lesser extent from the combination with HLA-I + HIVgp120 + HSP70 (4.7 ± 0.4 μg/ml and 92.8 ± 3.4%, respectively). Single or double antibody combination showed significantly higher IC50, from 6.5 to 19.0 μg/ml, and lower levels of HIV-1 inhibition. However, the decrease in IC50 was not synergistic or additive and the mechanism of the complementary neutralization activity needs to be elucidated. The proximity of the three molecules within the HIV-1 virion is not known, but it is likely that steric hindrance and conformational changes induced by the antibodies may favour neutralization. These results were not HLA-I or HLA-II allele specific, as comparable IC50 and dose-dependent inhibition was found with HLA-B27, B17, DR1, DR3 and DR4 antibodies. Thus, a combination of polyclonal human IgG antibodies with three diverse molecules expressed by HIV-1 virions are more effective, and in lower concentrations, than the corresponding single or double antibodies in inhibiting HIV-1 replication in human CD4 T cells.
To exclude other serum inhibitory factors and unrelated antibodies, mAb to the four molecules were studied. The results with the IgG mAb were consistent with those of polyclonal human IgG antibodies in that a progressive increase in dose-dependent inhibition was found with clade B HIV-1 by single, double or triple combination of mAb to HLA-I or HLA-II with HSP70 and HIV-1 gp120. The lowest IC50 of 3.5 (±1.3) μg/ml and maximum inhibition of 99 (±0.5)% resulted from a combination of the three mAbs to HLA-II + HIVgp120 + HSP70 and to a lesser extent from a combination of the mAbs to HLA-I + HIVgp120 + HSP70. Furthermore, a comparison of the broadly acting mAb b12 with the triple serum antibodies in the same assay showed an IC50 of 1.75 μg/ml with the mAb and 2.25 μg/ml with the antisera. Thus, the IC50 of the triple combination of antibodies compared favourably with the mAb b12, which is one of the ‘gold standards’ and has been very difficult to attain by conventional immunization.
To establish whether the combination of three polyclonal human antibodies elicit broadly neutralizing activity, further dose-dependent studies were pursued with two subtypes of HIV-1 clades A, C or D. These studies demonstrated that a combination of three human polyclonal antibodies to HLA-II with HIVgp120 and HSP70 elicited IC50 of 2.5–6.1 μg/ml and maximum inhibition of 75–100%, with the exception of clade A strains (92/RW/024) and clade C strain (93/IN/101). A direct comparison of the IC50 between antibodies to HIVgp120 and the triple antibody combination suggests that lower IC50 was achieved with the triple combination of antisera than the single gp120 antiserum with clades B, C and D (one strain each) but not with the two clade A strains and one clade C strain. These results should, however, be viewed in the context that the antisera to HLA and HSP70 were not from actively immunized persons, unlike the antisera to HIVgp120 that were from HIV-1-infected persons. Thus, broadly neutralizing antibody activity can be achieved with the combination of three polyclonal human antisera against three of the four clades of HIV-1 examined.
It is of interest that the antibody combinations failed to neutralize significantly CXCR4-using HIV-1 (LAI and group O). This was not studied further, but we can speculate that the polyclonal antibodies were most likely directed against CCR5-using viruses. HSP70 binds the CCR5 coreceptor  and inhibits infection mostly of R5 viruses , so although HSP70 antibodies would bind HSP70 both in the X4-type and R5-type HIV-1, the inhibitory effect would be seen only with the CCR5-using virus. Another aspect of HSP70 antibodies is that they are commonly found in normal human sera [38,39], can be increased on immunization and do not cause adverse effects.
The results of this investigation suggest that the trimolecular HLA–HIVgp120–HSP70 complex may constitute a novel vaccine strategy against HIV-1 infection, targeting not only the viral envelope gp120 but also defined host molecules expressed by the virus particles. The three components of such a vaccine have distinct functional properties. Alloimmune stimulation may engage up to 10% of T cells , elicit antibodies to HLA-II, which may bind CD4 molecules on T cells, macrophages and dendritic cells, and induce an array of cytokines and chemokines, which greatly enhance immunogenicity. HLA-I antibodies however may inhibit the cytotoxic function of CD8 T cells. HSP70 exerts mucosal and systemic adjuvanticities by stimulating chemokines, cytokines and maturation of dendritic cells [54,55]. HSP70 may also inhibit HIV-1 transmission by binding the CCR5 receptors [52,56], stimulating CC chemokines, which block CCR5, and upregulating the innate anti-HIV-1 APOBEC3G . HIVgp120 may elicit specific cellular and antibody responses to HIV-1, aided by the two HIV-1 non-cognate agents. Using the three antigens found in HIV virions is more likely to yield a more effective neutralizing antibody level than is possible by single antigen. This novel trimolecular vaccination strategy, utilizing HIV-1-specific and non-cognate immune responses, is unlikely to be subject to viral escape and will be examined in vivo in macaques to ascertain whether challenge with SHIV can be prevented or contained.
This work was supported partly by the European Community, Grants Empro (LSHP-CT-2003-503558) and Europrise (LSHP-CT-2006-037611), and by the Bill & Melinda Gates Foundation. We thank Dr P. Anton for the HIV-1 gp120 antibodies from HIV positive men and Dr R. Shattock for the PM1 cell line and clade O-HIV.
Dr K. Babaahmady was responsible for the neutralization studies with HIV-1, Dr L. Bergmeier carried out the screening of human sera for antibodies to HSP70 and separation of IgG from the human antisera. Dr T. Lehner conceived the project, was responsible for quality control and analyses of the data and wrote the paper with the aid of Drs Babaahmady and Bergmeier.
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HIV-1; HLA-I; HLA-II; neutralizing antibodies
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