Enhanced HIV-1 neutralization by antibody heteroligation

Passive transfer of broadly neutralizing human antibodies against HIV-1 protects macaques against infection. However, HIV-1 uses several strategies to escape antibody neutralization, including mutation of the gp160 viral surface spike, a glycan shield to block antibody access to the spike, and expression of a limited number of viral surface spikes, which interferes with bivalent antibody binding. The latter is thought to decrease antibody apparent affinity or avidity, thereby interfering with neutralizing activity. To test the idea that increasing apparent affinity might enhance neutralizing activity, we engineered bispecific anti-HIV-1 antibodies (BiAbs) that can bind bivalently by virtue of one scFv arm that binds to gp120 and a second arm to the gp41 subunit of gp160. The individual arms of the BiAbs preserved the binding specificities of the original anti-HIV IgG antibodies and together bound simultaneously to gp120 and gp41. Heterotypic bivalent binding enhanced neutralization compared with the parental antibodies. We conclude that antibody recognition and viral neutralization of HIV can be improved by heteroligation.     

A human monoclonal antibody neutralizes diverse HIV-1 isolates by binding a critical gp41 epitope

HIV-1 entry into cells is mediated by the envelope glycoprotein receptor-binding (gp120) and membrane fusion-promoting (gp41) subunits. The gp41 heptad repeat 1 (HR1) domain is the molecular target of the fusion-inhibitor drug enfuvirtide (T20). The HR1 sequence is highly conserved and therefore considered an attractive target for vaccine development, but it is unknown whether antibodies can access HR1. Herein, we use gp41-based peptides to select a human antibody, 5H/I1-BMV-D5 (D5), that binds to HR1 and inhibits the assembly of fusion intermediates in vitro. D5 inhibits the replication of diverse HIV-1 clinical isolates and therefore represents a previously unknown example of a crossneutralizing IgG selected by binding to designed antigens. NMR studies and functional analyses map the D5-binding site to a previously identified hydrophobic pocket situated in the HR1 groove. This hydrophobic pocket was proposed as a drug target and subsequently identified as a common binding site for peptide and peptidomimetic fusion inhibitors. The finding that the D5 fusion-inhibitory antibody shares the same binding site suggests that the hydrophobic pocket is a "hot spot" for fusion inhibition and an ideal target on which to focus a vaccine-elicited antibody response. Our data provide a structural framework for the design of new immunogens and therapeutic antibodies with crossneutralizing potential.     

HIV-1-specific antibodies in cerebrospinal fluid mediate cellular cytotoxicity and neutralization

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.     

An oligosaccharide-based HIV-1 2G12 mimotope vaccine induces carbohydrate-specific antibodies that fail to neutralize HIV-1 virions

The conserved oligomannose epitope, Man₉GlcNAc₂, recognized by the broadly neutralizing human mAb 2G12 is an attractive prophylactic vaccine candidate for the prevention of HIV-1 infection. We recently reported total chemical synthesis of a series of glycopeptides incorporating one to three copies of Man₉GlcNAc₂ coupled to a cyclic peptide scaffold. Surface plasmon resonance studies showed that divalent and trivalent, but not monovalent, compounds were capable of binding 2G12. To test the efficacy of the divalent glycopeptide as an immunogen capable of inducing a 2G12-like neutralizing antibody response, we covalently coupled the molecule to a powerful immune-stimulating protein carrier and evaluated immunogenicity of the conjugate in two animal species. We used a differential immunoassay to demonstrate induction of high levels of carbohydrate-specific antibodies; however, these antibodies showed poor recognition of recombinant gp160 and failed to neutralize a panel of viral isolates in entry-based neutralization assays. To ascertain whether antibodies produced during natural infection could recognize the mimetics, we screened a panel of HIV-1-positive and -negative sera for binding to gp120 and the synthetic antigens. We present evidence from both direct and competitive binding assays that no significant recognition of the glycopeptides was observed, although certain sera did contain antibodies that could compete with 2G12 for binding to recombinant gp120.     

Presence of HIV-1 neutralizing IgA antibodies in primary HIV-1 infected patients

The initial control of viral replication during primary HIV-1 infection is dominated by CD8+ T-cell mediated responses. Neutralizing IgG to autologous virus is first detected in serum weeks after this response when the viraemia has already declined. However, the mucosal and systemic HIV-1 neutralizing IgA response during primary HIV-1 infection in patients treated with HAART has not been studied previously. The presence of HIV-1 neutralizing IgA antibodies in serum (n=10 patients) and semen (n=6 patients) samples was tested against a laboratory adapted HIV-1 isolate and against primary HIV-1 isolates, representing different clades and phenotypes. The patients received HAART during the study period and were followed from primary HIV-1 infection and up to 72 weeks. Overall, HIV-1 neutralizing IgA activity could be demonstrated in serum from 5 of 10 primary HIV-1 infected patients at inclusion, although the response was restricted to only 1 of the 4 tested isolates. In semen samples, HIV-1 neutralizing IgA activity was seen in 2 of 5 patients against at least 1 of the HIV-1 isolates. In conclusion, a restricted but early neutralizing IgA response can be detected in serum and semen in primary infected patients treated with HAART.     

Kinetic mechanism for HIV-1 neutralization by antibody 2G12 entails reversible glycan binding that slows cell entry

Despite structural knowledge of broadly neutralizing monoclonal antibodies (NMAbs) complexed to HIV-1 gp120 and gp41 envelope glycoproteins, virus inactivation mechanisms have been difficult to prove, in part because neutralization assays are complex and were previously not understood. Concordant with recent evidence that HIV-1 titers are determined by a race between entry of cell-attached virions and competing inactivation processes, we show that NMAb 2G12, which binds to gp120 N-glycans with α (1, 2)-linked mannose termini and inhibits replication after passive transfer into patients, neutralizes by slowing entry of adsorbed virions. Accordingly, apparent neutralization is attenuated when a kinetically competing virus inactivation pathway is blocked. Moreover, removing 2G12 from media causes its dissociation from virions coupled to accelerated entry and restored infectivity, demonstrating the reversibility of neutralization. A difference between 2G12 dissociation and infectivity recovery rates implies that the inhibited complexes at virus-cell junctions contain several 2G12's that must dissociate before entry commences. Quantitative microscopy of 2G12 binding and dissociation from single virions and studies using a split CCR5 coreceptor suggest that 2G12 competitively inhibits interactions between gp120's V3 loop and the tyrosine sulfate-containing CCR5 amino terminus, thereby reducing assembly of complexes that catalyze entry. These results reveal a unique reversible kinetic mechanism for neutralization by an antibody that binds near a critical V3 region in the glycan shield of gp120.     

Broad neutralization and complement-mediated lysis of HIV-1 by PEHRG214, a novel caprine anti-HIV-1 polyclonal antibody

OBJECTIVES: To assess the potency, breadth of action, and mechanism of action of the polyclonal goat anti-HIV antibody, PEHRG214. DESIGN: Typical human antibody responses to HIV-1 infection are unable to neutralize virus efficiently, clear the infection, or prevent disease progression. However, more potent neutralizing antibodies may be capable of playing a pivotal role in controlling HIV replication in vivo. PEHRG214 is a polyclonal caprine antibody raised against purified HIV-associated proteins, such that epitopes that are immunologically silent in humans may potentially be recognized in another species. It has been administered safely to HIV-infected individuals in Phase I clinical trials. METHODS: The anti-HIV activity of PEHRG214 was assessed using neutralization and virion lysis assays. The target proteins for PEHRG214 activity were investigated using flow cytometry and by adsorption of anti-cell antibodies from the antibody cocktail. RESULTS: PEHRG214 strongly neutralized a diverse range of primary HIV-1 isolates, encompassing subtypes A to E and both CCR5 and CXCR4 phenotypes. Neutralization was enhanced by the presence of complement. PEHRG214 also induced complement-mediated lysis of all HIV-1 isolates tested, and recognized or cross-reacted with a number of host cell proteins. Lysis was abrogated by adsorption with T and/or B cells expressing GPI-linked proteins, but not by GPI-deficient B cells or red blood cells. CONCLUSIONS: PEHRG214 was found to potently neutralize and lyse HIV-1 particles. By targeting host cell proteins present in the viral envelope, which are conserved among all strains tested, PEHRG214 potentially opens up a highly novel means of eliminating circulating virus in infected individuals.     

Localization of epitopes for human factor VIII inhibitor antibodies by immunoblotting and antibody neutralization

Human factor VIII(FVIII) inhibitors are pathologic, circulating antibodies that inactivate FVIII. We have examined the location of epitopes on the FVIII protein for inhibitors from hemophilia A and nonhemophilic individuals. The inhibitors were of type I or type II in the kinetics of their inactivation of FVIII. A cDNA clone of human FVIII was used to express defined FVIII protein fragments in Escherichia coli for immunoblotting with inhibitor plasma. An epitope for 18 heavy-chain inhibitors was localized to the aminoterminal 18.3 Kd of the A2 domain. Two of these inhibitors also recognized an epitope located between A1 and A2 domains. Similarly, an epitope for 23 light- chain inhibitors was localized to the C2 domain. Weaker epitopes for 13 of the same inhibitors within the C1 and C2 domains were also observed. Four of the 23 inhibitors in addition bound strongly to the A3 domain. Most inhibitors (22 of 23) were neutralized in vitro only by the FVIII fragments to which they bound on immunoblots; however, one inhibitor that was neutralized by a fragment containing the A1 domain did not bind to it on immunoblots. Conversely, 3 of 3 inhibitors that bound to the A3 domain and 5 of 15 that bound to the A2 domain were not neutralized by the corresponding fragments. The epitope specificity of an inhibitor did not depend on its source or type. Our results show that FVIII inhibitors bind to limited areas within the heavy and light chains of FVIII. Some inhibitor plasmas contain additional antibodies that may not be inhibitory.     

Characterization of the specificity of the human antibody response to the V3 neutralization domain of HIV-1.

The major neutralization domain of HIV-1, contained in the third variable region (V3) of the external envelope, is highly variable at positions flanking a conserved glycine-proline-glycine sequence. We investigated the relation between V3 sequences of HIV-1 variants circulating in a host and that host's antibody specificity. Multiple V3 sequences were obtained directly, via PCR and subsequent cloning, from serum RNA or cellular DNA from 26 individuals (from 12 around seroconversion). Then, specificity of sera from these individuals to a panel of V3 peptides was determined. The specificity (best recognized peptide) of the early antibody response accurately reflected the virus population circulating around seroconversion in 12/12 individuals and 4/4 HIV-1-infected chimpanzees. A change in serum specificity at later stages of infection was rare: five years after seroconversion, only 3 of 46 individuals had a specificity that differed completely from that in the first year. However, the V3 domain of the virus does change over time, as evidenced by the poor correlation between V3 sequences obtained late in infection and V3 antibody reactivity at the same time point. Thus, in contrast to the accurate antibody response to HIV-1 variants early after infection, generally a specific response to variants emerging at later stages seemed to be absent or of low level. Instead, the early response appeared to be preserved. Finally, we made use of the observed accurate reflection to analyze the variation for the V3 domain of HIV-1 in the Netherlands by probing specificities of early sera from 129 Dutch seroconverting individuals. Specific reactivity to RKSIHIGPGRAFYTTG was found in 36%, to RKSINIGPGRAFYTTG in 12% and to RKSIPIGPGRAFYTTG in 18% of these Dutch sera.     

Characterization of the African HIV-1 isolate CBL-4 (RUT) by partial sequence analysis and virus neutralization with peptide antibody and antisense phosphate-methylated DNA

The HIV-1 isolate CBL-4 (RUT), originating from Tanzania, was characterized using a comprehensive virus-typing system. This system included sequence analysis of the region coding for the neutralization domain in the third variable region (V3) of the external envelope and of the tat responsive (TAR) region after polymerase chain reaction (PCR) amplification of these sequences from cellular DNA in the CBL-4 (RUT) producer line. Based on independent cluster analysis of TAR and V3 sequences the CBL-4 (RUT) virus was positioned closest to the Z6 (and ELI) African virus family. The V3 amino acid sequence on the surface of the virus particle was confirmed by the inhibition of neutralization of CBL-4 (RUT) by a synthetic peptide derived from the nucleic acid sequence. Using antisense phosphate-methylated DNA covering the TAR loop region of LAV-1/HTLV-IIIB, inhibition of HTLV-IIIB and HTLV-IIIRF infection was seen, whereas no inhibition was observed for CBL-4 (RUT), indicating two or more mismatches in the TAR loop region, a characteristic shared with Z6 virus, but not with ELI. We propose a virus-typing system based on sequence analysis confirmed by virus neutralization with a peptide binding antibody and inhibition by antisense phosphate-methylated DNA to group viruses for laboratory use and vaccine design.     

Cross-clade HIV-1 neutralization by an antibody fragment from a lupus phage display library

A single-chain fragment containing antibody V domains (scFv) isolated from a lupus antibody library displayed the ability to bind gp120 and the conserved gp120 determinant composed of residues 421-436. The scFv neutralized R5 and X4-dependent HIV-1 strains from clades B, C, and D. The lupus repertoire may be useful as a source of neutralizing antibodies to HIV.     

Characterization of HIV-1 neutralization escape mutants

Infection by molecularly cloned HIV-1, in the presence of a high-titre neutralizing monoclonal antibody (MAb), resulted in the selection of plaques in MT4 cells releasing HIV resistant to neutralization by the same MAb. The epitope recognized by the MAb was mapped to the V3 neutralization epitope at amino acids 305-321. The HIV-1 variants showed a reduced binding capacity for the selecting MAb as determined by immunofluorescence. Polymerase chain reaction (PCR) amplification of complementary DNA derived from viral RNA, cloning and sequencing identified a base pair (bp) change C----G at position 6663 in variant 110.5/1, predicting a change at amino acid 308 Arg----Gly. No other changes in the epitope were observed by sequencing three other variants. Differential hybridization of PCR amplified viral RNA and DNA, with oligonucleotides specific for the observed bp change or the 'wild type' sequence, indicated that the variants 110.5/1 and 110.5/7 were genotypically mixed for 308Gly/Arg. Subsequent screening of biologically 'recloned' variants 110.5/1 and 110.5/7 identified two subclones homozygous for the 308Gly change. The Arg----Gly change appears to affect the binding of the antibody to the epitope, since the linear peptide substituting 308Gly for 'wild type' 308Arg was 100 times less potent in blocking the neutralization of parental HIV. Amino-acid residue 308 thus appears to be crucial for antibody binding to the epitope. In addition, mutations distant from the monoclonal antibody binding site may also affect neutralization by antibodies recognizing the V3 loop.     

Rapid development of isolate-specific neutralizing antibodies after primary HIV-1 infection and consequent emergence of virus variants which resist neutralization by autologous sera

The kinetics of appearance and specificity of HIV-1 neutralizing antibodies was studied in four individuals. HIV-1 was isolated during symptomatic primary HIV-1 infection and repeatedly thereafter, and tested against autologous sera collected in parallel. Our patients developed isolate-specific low-titer neutralizing antibodies within 2-4 weeks, and the titers to the first isolates increased with time. We documented the emergence of virus variants with reduced sensitivity to neutralization by autologous, but not heterologous, sera in three patients. These virus variants were not, however, resistant to neutralization per se, since they were readily neutralized by the positive control serum. Our patients did not develop antibodies capable of neutralizing the new virus variants during the observation period. This suggests either a failure of the immune system to respond to the new virus variants or a mechanism by which the virus evades detection by the immune system. The emergence of neutralization-resistant virus variants was not directly correlated with disease progression since two patients have remained asymptomatic after the emergence of such virus variants. It is, however, likely that the emergence of virus variants which the patient fails to neutralize in the long run contributes to disease progression.     

The role of the third β strand in gp120 conformation and neutralization sensitivity of the HIV-1 primary isolate DH012

Neutralization of HIV-1 primary isolates has been a tremendous challenge for AIDS vaccine development. Here, we identify a single amino acid change (T198P) in gp120 that alters the neutralization sensitivity of the primary isolate DH012 to antibodies against multiple neutralization epitopes that include the V3, CD4-induced, and CD4 binding sites in gp120. This mutation is located in the V1/V2 stem region that forms the third beta strand (beta3) of the bridging sheet of gp120. The conformation of variable loops, especially V1/V2 and V3, was proposed to regulate the accessibility of these neutralization epitopes. The results of this study indicate a direct association between the V1/V2 and V3 loops of DH012 gp120. The single amino acid mutation T198P in the beta3 severely compromises the interaction between the V1/V2 and V3 loops. These results suggest that interaction of V1/V2 and V3 can mask the neutralization epitopes and that the beta3 plays a critical role in determining the neutralization sensitivity by modulating the interaction. This study provides an insight into why primary isolates are relatively resistant to antibody neutralization and might facilitate the development of anti-HIV strategies against HIV-1 infection.     

Thermodynamic and kinetic analysis of sCD4 binding to HIV-1 virions and of gp120 dissociation.

Kinetic and thermodynamic aspects of the binding of sCD4 to intact virions of human immunodeficiency virus type 1 (HIV-1 RF), and of the subsequent induction of gp120 dissociation were studied. sCD4 binding to virions at 4 and 37 degrees C is half-maximal at approximately 40 and 10 nM, respectively. The transition between low-affinity and high-affinity binding of sCD4 to virions occurs over a narrow temperature range between 20 and 25 degrees C. Shedding of gp120 from virions after sCD4 binding is also temperature dependent, being initiated above approximately 20 degrees C. The minimum temperatures for the sCD4 affinity transition and gp120 shedding are, therefore, similar and we suggest how the two processes might be related mechanistically.     

Mucosal and plasma IgA from HIV-exposed seronegative individuals neutralize a primary HIV-1 isolate

OBJECTIVE: To characterize functional properties of HIV-specific IgA in samples representing both systemic and mucosal compartments of HIV-1 highly exposed persistently seronegative (HEPS) individuals. METHODS: IgA was purified from plasma and mucosal samples from HEPS individuals and tested for the ability to neutralize infection of peripheral blood mononuclear cells (PBMC) by a non-syncytium inducing HIV-1 (clade B) primary isolate. None of these individuals had measurable HIV-1-specific IgG. RESULTS: HIV-1-specific neutralizing activity of the purified IgA from plasma (n = 15), saliva (n = 15) and cervicovaginal fluid (CVF) (n = 14) were found in the majority of samples (73, 73 and 79%, respectively). In contrast, plasma, saliva and CVF samples of low-risk, uninfected HIV-seronegative individuals lacked neutralizing IgA, with the exception of two out of 34 (6%) saliva samples. CONCLUSION: Mucosal and plasma IgA from HEPS individuals can neutralize HIV-1 infection.     

Neutralizing antibodies against two HIV-1 strains in consecutively collected serum samples: cross neutralization and association to HIV-1 related disease.

97 sera collected during a 10-year period from 10 HIV-1 infected individuals were tested for neutralizing capacity against a virus isolate FICPH-22 obtained from a Danish AIDS patient, and the laboratory strain HTLV-IIIB. Three patterns of serum neutralizing activity were demonstrated: (a) patients developing high neutralizing activity against both HIV strains; (b) patients developing high neutralizing activity against the Danish virus isolate; and (c) patients developing only low titers of neutralizing antibodies (NA) against both HIV strains. The HTLV-IIIB strain was less sensitive to serum neutralization than the FICPH-22 isolate and the appearance of NA against HTLV-IIIB was typically lacking several years behind that against FICPH-22 indicating a broadening of the NA response over time. No difference in clinical outcome was observed comparing patients reaching high titers of NA and patients with low titers. Development of AIDS among patients reaching high titers of NA was preceded by a decline in NA titers, indicating an association of high titers of NA with the healthy carrier state and of declining or low titers of NA with disease progression. The majority of the neutralizing activity was mediated by IgG, but some neutralizing activity was demonstrated in the IgG depleted serum, indicating the presence of additional neutralizing substances in serum.