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1.  Comparative Magnitude of Cross-Strain Conservation of HIV Variable Loop Neutralization Epitopes 
PLoS ONE  2010;5(12):e15994.
Although the sequence variable loops of the human immunodeficiency virus' (HIV-1) surface envelope glycoprotein (gp120) can exhibit good immunogenicity, characterizing conserved (invariant) cross-strain neutralization epitopes within these loops has proven difficult. We recently developed a method to derive sensitive and specific signature motifs for the three-dimensional (3D) shapes of the HIV-1 neutralization epitopes in the third variable (V3) loop of gp120 that are recognized by human monoclonal antibodies (mAbs). We used the signature motif method to estimate the conservation of these epitopes across circulating worldwide HIV-1 strains. The epitope targeted by the anti-V3 loop neutralizing mAb 3074 is present in 87% of circulating strains, distributed nearly evenly among all subtypes. The results for other anti-V3 Abs are: 3791, present in 63% of primarily non-B subtypes; 2219, present in 56% of strains across all subtypes; 2557, present in 52% across all subtypes; 447-52D, present in 11% of primarily subtype B strains; 537-10D, present in 9% of primarily subtype B strains; and 268-D, present in 5% of primarily subtype B strains. The estimates correlate with in vitro tests of these mAbs against diverse viral panels. The mAb 3074 thus targets an epitope that is nearly completely conserved among circulating HIV-1 strains, demonstrating the presence of an invariant structure hidden in the dynamic and sequence-variable V3 loop in gp120. Since some variable loop regions are naturally immunogenic, designing immunogens to mimic their conserved epitopes may be a promising vaccine discovery approach. Our results suggest one way to quantify and compare the magnitude of the conservation.
PMCID: PMC3012121  PMID: 21209919
2.  Structural Basis of HIV-1 Neutralization by Affinity Matured Fabs Directed against the Internal Trimeric Coiled-Coil of gp41 
PLoS Pathogens  2010;6(11):e1001182.
The conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 is transiently exposed during the fusion process by forming a pre-hairpin intermediate, thus representing an attractive target for the design of fusion inhibitors and neutralizing antibodies. In previous studies we reported a series of broadly neutralizing mini-antibodies derived from a synthetic naïve human combinatorial antibody library by panning against a mimetic of the trimeric N-HR coiled coil, followed by affinity maturation using targeted diversification of the CDR-H2 loop. Here we report crystal structures of the N-HR mimetic 5-Helix with two Fabs that represent the extremes of this series: Fab 8066 is broadly neutralizing across a wide panel of B and C type HIV-1 viruses, whereas Fab 8062 is non-neutralizing. The crystal structures reveal important differences in the conformations of the CDR-H2 loops in the complexes that propagate into other regions of the antigen-antibody interface, and suggest that both neutralization properties and affinity for the target can be attributed, at least in part, to the differences in the interactions of the CDR-H2 loops with the antigen. Furthermore, modeling of the complex of an N-HR trimer with three Fabs suggests that the CDR-H2 loop may be involved in close intermolecular contacts between neighboring antibody molecules, and that such contacts may hinder the formation of complexes between the N-HR trimer and more than one antibody molecule depending on the conformation of the bound CDR-H2 loop which is defined by its interactions with antigen. Comparison with the crystal structure of the complex of 5-Helix with another neutralizing monoclonal antibody known as D5, derived using an entirely different antibody library and panning procedure, reveals remarkable convergence in the optimal sequence and conformation of the CDR-H2 loop.
Author Summary
Membrane fusion of HIV-1 with its target cells represents the first step in viral infection. This process involves a series of conformational changes in two viral envelope glycoproteins, gp120 and gp41, subsequent to binding of gp120 to the CD4 receptor and the chemokine coreceptor on the target cell membrane. During the fusion process, the conserved N-heptad repeat (N-HR) of gp41 in the form of a trimeric coiled-coil is accessible and presents an attractive target for the generation of broadly neutralizing antibodies. Here we present the crystal structures of two monoclonal Fabs complexed to a mimetic of the N-HR trimer. These Fabs were derived from a synthetic human combinatorial antibody library comprising more than 1010 human specificities by first panning against an N-HR mimetic, followed by affinity maturation through targeted diversification of the CDR-H2 complementarity determining region. One of the Fabs is broadly neutralizing across a wide range of primary isolates from subtype B and C HIV-1, whereas the other one is non-neutralizing. Our structures reveal the key role of the CDR-H2 loop in antigen recognition and how this correlates with HIV-1 neutralization properties.
PMCID: PMC2978731  PMID: 21085615
3.  Discrimination of Subtype B and Non-Subtype B Strains of Human Immunodeficiency Virus Type 1 by Serotyping: Correlation with Genotyping 
Journal of Clinical Microbiology  1999;37(5):1356-1360.
The ability of a peptide-based serotyping assay to differentiate human immunodeficiency virus (HIV) type 1 (HIV-1) subtype B infections from non-subtype B infections was investigated with 166 anti-HIV-1- and HIV RNA-positive (by PCR) serum or plasma specimens. The specimens were divided genetically into those infected with subtype B and non-subtype B by application of a screening heteroduplex mobility assay (HMA) that used plasmids for subtypes A and B alone. Specimens that were not clearly infected with HIV-1 subtype B by HMA or for which the two methods had discordant results in distinguishing those infected with subtype B from those infected with non-subtype B were then investigated with a full HMA plasmid panel and, for selected specimens, env sequencing. For the 141 genotyped and serotypically reactive specimens, the correlation between genotyping and serotyping (all subtypes) was 69%. Of the 67 specimens that reacted monotypically as serotype B, 64 were shown to be infected with genotype B (positive predictive value, 96%). Of the 82 specimens that contained genotype B nucleic acid, 64 reacted monotypically as serotype B (sensitivity, 78%), and 4 specimens reacted with a single non-subtype B peptide; the viruses in 14 specimens could not be assigned a serotype. Initial screening results had indicated that 12 samples had results discordant between restricted HMA and serotyping. The V3 loop amino acids of the infecting HIV strains from the seven specimens with discordant serology results were analyzed. For five specimens discordance occurred when the amino acid sequence of the infecting virus closely resembled those of more than one consensus peptide antigen or when the observed V3 crown motif of the strain was atypical for the genetic subtype present. For the other two specimens no explanation for the discordance was identified. Five specimens gave unclear or discordant results in the initial HMA screen, but the results were resolved when the full plasmid panel was used. Serotyping, although of limited sensitivity, distinguishes between subtype B and non-subtype B infections with a high degree of specificity. However, it poorly differentiates the major non-subtype B subtypes, particularly subtypes A and C. When HIV-1 subtype B predominates, serological typing and/or subtype-restricted HMA screening usefully distinguishes between subtype B and non-subtype B infections.
PMCID: PMC84775  PMID: 10203486
4.  Anti-V3 Monoclonal Antibodies Display Broad Neutralizing Activities against Multiple HIV-1 Subtypes 
PLoS ONE  2010;5(4):e10254.
The V3 loop of the HIV-1 envelope (Env) glycoprotein gp120 was identified as the “principal neutralizing domain” of HIV-1, but has been considered too variable to serve as a neutralizing antibody (Ab) target. Structural and immunochemical data suggest, however, that V3 contains conserved elements which explain its role in binding to virus co-receptors despite its sequence variability. Despite this evidence of V3 conservation, the ability of anti-V3 Abs to neutralize a significant proportion of HIV-1 isolates from different subtypes (clades) has remained controversial.
HIV-1 neutralization experiments were conducted in two independent laboratories to test human anti-V3 monoclonal Abs (mAbs) against pseudoviruses (psVs) expressing Envs of diverse HIV-1 subtypes from subjects with acute and chronic infections. Neutralization was defined by 50% inhibitory concentrations (IC50), and was statistically assessed based on the area under the neutralization titration curves (AUC).
Using AUC analyses, statistically significant neutralization was observed by ≥1 anti-V3 mAbs against 56/98 (57%) psVs expressing Envs of diverse subtypes, including subtypes A, AG, B, C and D. Even when the 10 Tier 1 psVs tested were excluded from the analysis, significant neutralization was detected by ≥1 anti-V3 mAbs against 46/88 (52%) psVs from diverse HIV-1 subtypes. Furthermore, 9/24 (37.5%) Tier 2 viruses from the clade B and C standard reference panels were neutralized by ≥1 anti-V3 mAbs. Each anti-V3 mAb tested was able to neutralize 28–42% of the psVs tested. By IC50 criteria, 40/98 (41%) psVs were neutralized by ≥1 anti-V3 mAbs.
Using standard and new statistical methods of data analysis, 6/7 anti-V3 human mAbs displayed cross-clade neutralizing activity and revealed that a significant proportion of viruses can be neutralized by anti-V3 Abs. The new statistical method for analysis of neutralization data provides many advantages to previously used analyses.
PMCID: PMC2858080  PMID: 20421997
5.  Enhancing Exposure of HIV-1 Neutralization Epitopes through Mutations in gp41 
PLoS Medicine  2008;5(1):e9.
The generation of broadly neutralizing antibodies is a priority in the design of vaccines against HIV-1. Unfortunately, most antibodies to HIV-1 are narrow in their specificity, and a basic understanding of how to develop antibodies with broad neutralizing activity is needed. Designing methods to target antibodies to conserved HIV-1 epitopes may allow for the generation of broadly neutralizing antibodies and aid the global fight against AIDS by providing new approaches to block HIV-1 infection. Using a naturally occurring HIV-1 Envelope (Env) variant as a template, we sought to identify features of Env that would enhance exposure of conserved HIV-1 epitopes.
Methods and Findings
Within a cohort study of high-risk women in Mombasa, Kenya, we previously identified a subtype A HIV-1 Env variant in one participant that was unusually sensitive to neutralization. Using site-directed mutagenesis, the unusual neutralization sensitivity of this variant was mapped to two amino acid mutations within conserved sites in the transmembrane subunit (gp41) of the HIV-1 Env protein. These two mutations, when introduced into a neutralization-resistant variant from the same participant, resulted in 3- to >360-fold enhanced neutralization by monoclonal antibodies specific for conserved regions of both gp41 and the Env surface subunit, gp120, >780-fold enhanced neutralization by soluble CD4, and >35-fold enhanced neutralization by the antibodies found within a pool of plasmas from unrelated individuals. Enhanced neutralization sensitivity was not explained by differences in Env infectivity, Env concentration, Env shedding, or apparent differences in fusion kinetics. Furthermore, introduction of these mutations into unrelated viral Env sequences, including those from both another subtype A variant and a subtype B variant, resulted in enhanced neutralization susceptibility to gp41- and gp120-specific antibodies, and to plasma antibodies. This enhanced neutralization sensitivity exceeded 1,000-fold in several cases.
Two amino acid mutations within gp41 were identified that expose multiple discontinuous neutralization epitopes on diverse HIV-1 Env proteins. These exposed epitopes were shielded on the unmodified viral Env proteins, and several of the exposed epitopes encompass desired target regions for protective antibodies. Env proteins containing these modifications could act as a scaffold for presentation of such conserved domains, and may aid in developing methods to target antibodies to such regions.
Julie Overbaugh and colleagues analyze an HIV strain with high susceptibility to antibody neutralization and identify two gp41 envelope mutations that confer this sensitivity by exposing multiple neutralization epitopes.
Editors' Summary
In 1984 when scientists identified human immunodeficiency virus (HIV)—the cause of acquired immunodeficiency syndrome (AIDS)—many experts believed that a vaccine against HIV infection would soon be developed. Nearly 25 years later, there is still no such vaccine and with about 2.5 million new HIV infections in 2007, an effective vaccine is urgently needed to contain the AIDS epidemic. Vaccines provide protection against infectious diseases by priming the immune system to deal quickly and effectively with viruses and other pathogens. Vaccines do this by exposing the immune system to an immunogen—a fragment or harmless version of the pathogen. The immune system mounts a response against the immunogen and also “learns” from this experience so that if it is ever challenged with a virulent version of the same pathogen, it can quickly contain the threat. Many vaccines work by stimulating an antibody response. Antibodies are proteins made by the immune system that bind to molecules called antigens on the surface of pathogens. Antibodies that inactivate the invader upon binding to it are called “neutralizing” antibodies.
Why Was This Study Done?
Several characteristics of HIV have hampered the development of an effective vaccine. An “envelope” protein consisting of two subunits called gp120 and gp41 covers the outside of HIV. Many regions of this protein change rapidly, so the antibody response stimulated by a vaccine containing the envelope protein of one HIV variant provides little protection against other variants. However, other regions of the protein rarely change, so a vaccine that stimulates the production of antibodies to these “conserved” regions is likely to provide protection against many HIV variants. That is, it will stimulate the production of broadly neutralizing antibodies. Unfortunately, it has been difficult to find HIV vaccines that do this, because these conserved regions are often hidden from the immune system by other parts of the envelope protein. In this study, the researchers investigate the envelope protein of an HIV-1 variant they have isolated that is highly susceptible to inactivation by antibodies specific for these conserved regions. Comparing the envelope protein of this sensitive virus to closely related envelope proteins that are resistant to neutralization could identify features that might, if included in an envelope protein immunogen, produce a vaccine capable of generating broadly neutralizing antibodies.
What Did the Researchers Do and Find?
The researchers isolated a subtype A HIV-1 variant from a newly infected woman in Kenya that was efficiently neutralized by monoclonal antibodies (antibodies made by cells that have been cloned in the laboratory). These antibodies were specific for several different conserved regions of gp41 and gp120. The isolate was also neutralized by antibodies in blood from HIV-1-infected people. The envelope protein of the sensitive variant was the same as that of a resistant variant isolated at the same time from the woman, except for four amino acid changes in conserved regions of gp41 (proteins are made from long strings of amino acids). Using a technique called site-directed mutagenesis, the researchers introduced these amino acid changes into envelope proteins made in the laboratory and determined that just two of these changes were responsible for the neutralization sensitivity of the HIV-1 variant. The introduction of these two changes into the neutralization resistant variant and into the unrelated envelope sequences of another subtype A (common in Africa) HIV-1 variant and a subtype B HIV-1 (common in Europe and the Western Hemisphere) variant increased the sensitivity of all these viruses to antibody neutralization.
What Do These Findings Mean?
These findings show that two amino acid changes in gp41 of a neutralization-sensitive HIV-1 variant are responsible for the sensitivity of this variant to several neutralizing antibodies. The finding that the inclusion of these changes in the envelope protein of neutralization-resistant HIV-1 variants greatly increases their sensitivity to neutralizing antibodies indicates that the normally shielded regions of the protein are somehow made accessible to antibody by these changes. One possibility is that the amino acid changes might modify the overall shape of the envelope protein, thus exposing multiple, normally hidden regions in the HIV-1 envelope protein to antibodies. Importantly, these findings open up the possibility that the inclusion of these modifications in envelope-based immunogens might improve the ability of vaccines to generate broadly neutralizing antibodies against HIV-1.
Additional Information.
Please access these Web sites via the online version of this summary at
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS
HIVInSite has comprehensive information on all aspects of HIV/AIDS, including links to resources dealing with HIV vaccine development
Information is available from Avert, an international AIDS charity, on all aspects of HIV and AIDS, including HIV vaccines
The US Centers for Disease Control and prevention provides information on HIV/AIDS including information on its HIV vaccine unit (in English and some information in Spanish)
The AIDS Vaccine Clearinghouse provides clear information about HIV vaccine science, research and product development
The International AIDS Vaccine Initiative also provides straightforward information about the development of HIV vaccines
PMCID: PMC2174964  PMID: 18177204
6.  Production and characterization of human anti-V3 monoclonal antibodies from the cells of HIV-1 infected Indian donors 
Virology Journal  2012;9:196.
Analysis of human monoclonal antibodies (mAbs) developed from HIV-1 infected donors have enormously contributed to the identification of neutralization sensitive epitopes on the HIV-1 envelope glycoprotein. The third variable region (V3) is a crucial target on gp120, primarily due to its involvement in co-receptor (CXCR4 or CCR5) binding and presence of epitopes recognized by broadly neutralizing antibodies.
Thirty-three HIV-1 seropositive drug naive patients (18 males and 15 females) within the age range of 20–57 years (median = 33 years) were recruited in this study for mAb production. The mAbs were selected from EBV transformed cultures with conformationally constrained Cholera-toxin-B containing V3C (V3C-CTB) fusion protein. We tested the mAbs for their binding with HIV-1 derived proteins and peptides by ELISA and for neutralization against HIV-1 viruses by TZM-bl assays.
We isolated three anti-V3 mAbs, 277, 903 and 904 from the cells of different individuals. The ELISA binding revealed a subtype-C and subtype-A specific binding of antibody 277 and 903 while mAb 904 exhibited cross reactivity also with subtype-B V3. Epitope mapping of mAbs with overlapping V3 peptides showed exclusive binding to V3 crown. The antibodies displayed high and low neutralizing activity against 2/5 tier 1 and 1/6 tier 2 viruses respectively. Overall, we observed a resistance of the tier 2 viruses to neutralization by the anti-V3 mAbs, despite the exposure of the epitopes recognized by these antibodies on two representative native viruses (Du156.12 and JRFL), suggesting that the affinity of mAb might equally be crucial for neutralization, as the epitope recognition.
Our study suggests that the anti-V3 antibodies derived from subtype-C infected Indian patients display neutralization potential against tier 1 viruses while such activity may be limited against more resistant tier 2 viruses. Defining the fine epitope specificities of these mAbs and further experimental manipulations will be helpful in identification of epitopes, unique to clade C or shared with non-clade C viruses, in context of V3 region.
PMCID: PMC3493341  PMID: 22971578
HIV-1; Envelope glycoprotein; Third variable region; Anti-V3 monoclonal antibodies; Viral neutralization
7.  Worldwide Distribution of HIV Type 1 Epitopes Recognized by Human Anti-V3 Monoclonal Antibodies 
Epitopes, also known as antigenic determinants, are small clusters of specific atoms within macromolecules that are recognized by the immune system. Such epitopes can be targeted with vaccines designed to protect against specific pathogens. The third variable loop (V3 loop) of the HIV-1 pathogen's gp120 surface envelope glycoprotein can be a highly sensitive neutralization target. We derived sequence motifs for the V3 loop epitopes recognized by the human monoclonal antibodies (mAbs) 447-52D and 2219. Searching the HIV database for the occurrence of each epitope motif in worldwide viruses and correcting the results based on published WHO epidemiology reveal that the 447-52D epitope we defined occurs in 13% of viruses infecting patients worldwide: 79% of subtype B viruses, 1% of subtype C viruses, and 7% of subtype A/AG sequences. In contrast, the epitope we characterized for human anti-V3 mAb 2219 is present in 30% of worldwide isolates but is evenly distributed across the known HIV-1 subtypes: 48% of subtype B strains, 40% of subtype C, and 18% of subtype A/AG. Various assays confirmed that the epitopes corresponding to these motifs, when expressed in the SF162 Env backbone, were sensitively and specifically neutralized by the respective mAbs. The method described here is capable of accurately determining the worldwide occurrence and subtype distribution of any crystallographically resolved HIV-1 epitope recognized by a neutralizing antibody, which could be useful for multivalent vaccine design. More importantly, these calculations demonstrate that globally relevant, structurally conserved epitopes are present in the sequence variable V3 loop.
PMCID: PMC2853842  PMID: 19320565
8.  Factors Determining the Breadth and Potency of Neutralization by V3-Specific Human Monoclonal Antibodies Derived from Subjects Infected with Clade A or Clade B Strains of Human Immunodeficiency Virus Type 1 
Journal of Virology  2006;80(14):7127-7135.
The neutralizing activities of anti-V3 antibodies for HIV-1 isolates is affected both by sequence variation within V3 and by epitope masking by the V1/V2 domain. To analyze the relative contribution of V3 sequence variation, chimeric Env genes that contained consensus V3 sequences from seven HIV-1 subtypes in the neutralization-sensitive SF162 Env backbone were constructed. Resulting viral pseudotypes were tested for neutralization by 15 anti-V3 MAbs isolated from humans infected with viruses of either subtype B (anti-V3B MAbs) or subtype A (anti-V3A MAbs). Pseudovirions with the subtype B consensus V3 sequence were potently neutralized (IC50 < 0.006 μg/ml) by all but one of these MAbs, while pseudovirions with V3 subtypes A, C, F, H, AG, and AE were generally neutralized more effectively by anti-V3A MAbs than by anti-V3B MAbs. A V1/V2-masked Env version of SF162 Env with the consensus B V3 sequence was also neutralized by these MAbs, although with considerably lower potency, while similarly masked chimeras with V3 sequences of subtype A, C, or AG were weakly neutralized by anti-V3A MAbs but not by anti-V3B MAbs. Mutations in the V1/V2 domain of YU-2 Env increased the sensitivity of this highly resistant Env to a pool of anti-V3B MAbs several thousand-fold. These results demonstrated (i) the exceptional sensitivity of representative V3 domains of multiple subtypes to neutralization in the absence of epitope masking, (ii) the broader neutralizing activity of anti-V3A MAbs for viruses containing diverse V3 sequences, and (iii) the generality and dominant effect of V1/V2 masking on restriction of V3-mediated neutralization.
PMCID: PMC1489036  PMID: 16809318
9.  V3 Loop Truncations in HIV-1 Envelope Impart Resistance to Coreceptor Inhibitors and Enhanced Sensitivity to Neutralizing Antibodies 
PLoS Pathogens  2007;3(8):e117.
The V1/V2 region and the V3 loop of the human immunodeficiency virus type I (HIV-1) envelope (Env) protein are targets for neutralizing antibodies and also play an important functional role, with the V3 loop largely determining whether a virus uses CCR5 (R5), CXCR4 (X4), or either coreceptor (R5X4) to infect cells. While the sequence of V3 is variable, its length is highly conserved. Structural studies indicate that V3 length may be important for interactions with the extracellular loops of the coreceptor. Consistent with this view, genetic truncation of the V3 loop is typically associated with loss of Env function. We removed approximately one-half of the V3 loop from three different HIV-1 strains, and found that only the Env protein from the R5X4 strain R3A retained some fusion activity. Loss of V1/V2 (ΔV1/V2) was well tolerated by this virus. Passaging of virus with the truncated V3 loop resulted in the derivation of a virus strain that replicated with wild-type kinetics. This virus, termed TA1, retained the V3 loop truncation and acquired several adaptive changes in gp120 and gp41. TA1 could use CCR5 but not CXCR4 to infect cells, and was extremely sensitive to neutralization by HIV-1 positive human sera, and by antibodies to the CD4 binding site and to CD4-induced epitopes in the bridging sheet region of gp120. In addition, TA1 was completely resistant to CCR5 inhibitors, and was more dependent upon the N-terminal domain of CCR5, a region of the receptor that is thought to contact the bridging sheet of gp120 and the base of the V3 loop, and whose conformation may not be greatly affected by CCR5 inhibitors. These studies suggest that the V3 loop protects HIV from neutralization by antibodies prevalent in infected humans, that CCR5 inhibitors likely act by disrupting interactions between the V3 loop and the coreceptor, and that altered use of CCR5 by HIV-1 associated with increased sensitivity to changes in the N-terminal domain can be linked to high levels of resistance to these antiviral compounds.
Author Summary
The envelope protein of HIV-1 is responsible for binding virus to the surface of cells and mediating viral entry. Viral entry can be prevented by neutralizing antibodies that bind to envelope, and by small molecule inhibitors that bind to viral receptors on the cell surface, such as CCR5. HIV may acquire resistance to these small molecule inhibitors, several of which are being used in clinical trials to treat HIV-infected individuals, through resistance mechanisms that are not well understood. In addition, broadly neutralizing antibodies are rare—the envelope protein possesses structural features that limit antibody binding. We made a partial deletion in a region of envelope that interacts with viral receptors, and which is also widely believed to act as a shield against neutralizing antibodies. Normally, an envelope with such a modification would have total loss of function. However, by passaging virus with the partially deleted envelope in vitro, the envelope acquired adaptive mutations that restored function. Virus with the adapted envelope was highly sensitive to neutralizing antibodies and so may serve as a platform for immunization. This envelope also exhibited complete resistance to small molecule inhibitors that bind to the viral receptor CCR5, and lends insight into a mechanism of drug resistance by which the virus interacts with viral receptors on the cell surface in a novel manner.
PMCID: PMC1950945  PMID: 17722977
10.  Human Monoclonal Antibodies Specific for Conformation-Sensitive Epitopes of V3 Neutralize Human Immunodeficiency Virus Type 1 Primary Isolates from Various Clades 
Journal of Virology  2002;76(18):9035-9045.
The epitopes of the V3 domain of the human immunodeficiency virus type 1 (HIV-1) gp120 glycoprotein have complex structures consisting of linear and conformational antigenic determinants. Anti-V3 antibodies (Abs) recognize both types of elements, but Abs which preferentially react to the conformational aspect of the epitopes may have more potent neutralizing activity against HIV-1, as recently suggested. To test this hypothesis, human anti-V3 monoclonal Abs (MAbs) were selected using a V3 fusion protein (V3-FP) which retains the conformation of the third variable region. The V3-FP consists of the V3JR-CSF sequence inserted into a truncated form of murine leukemia virus gp70. Six human MAbs which recognize epitopes at the crown of the V3 loop were selected with the V3-FP. They were found to react more strongly with molecules displaying conformationally intact V3 than with linear V3 peptides. In a virus capture assay, these MAbs showed cross-clade binding to native, intact virions of clades A, B, C, D, and F. No binding was found to isolates from subtype E. The neutralizing activity of MAbs against primary isolates was determined in three assays: the GHOST cell assay, a phytohemagglutinin-stimulated peripheral blood mononuclear cell assay, and a luciferase assay. While these new MAbs displayed various degrees of activity, the pattern of cross-clade neutralization of clades A, B, and F was most pronounced. The neutralization of clades C and D viruses was weak and sporadic, and neutralization of clade E by these MAbs was not detected. Analysis by linear regression showed a highly significant correlation (P < 0.0001) between the strength of binding of these anti-V3 MAbs to intact virions and the percent neutralization. These studies demonstrate that human MAbs to conformation-sensitive epitopes of V3 display cross-clade reactivity in both binding to native, intact virions and neutralization of primary isolates.
PMCID: PMC136433  PMID: 12186887
11.  Cryptic Nature of a Conserved, CD4-Inducible V3 Loop Neutralization Epitope in the Native Envelope Glycoprotein Oligomer of CCR5-Restricted, but Not CXCR4-Using, Primary Human Immunodeficiency Virus Type 1 Strains 
Journal of Virology  2005;79(11):6957-6968.
The external subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env), gp120, contains conserved regions that mediate sequential interactions with two cellular receptor molecules, CD4 and a chemokine receptor, most commonly CCR5 or CXCR4. However, antibody accessibility to such regions is hindered by diverse protective mechanisms, including shielding by variable loops, conformational flexibility and extensive glycosylation. For the conserved neutralization epitopes hitherto described, antibody accessibility is reportedly unrelated to the viral coreceptor usage phenotype. Here, we characterize a novel, conserved gp120 neutralization epitope, recognized by a murine monoclonal antibody (MAb), D19, which is differentially accessible in the native HIV-1 Env according to its coreceptor specificity. The D19 epitope is contained within the third variable (V3) domain of gp120 and is distinct from those recognized by other V3-specific MAbs. To study the reactivity of MAb D19 with the native oligomeric Env, we generated a panel of PM1 cells persistently infected with diverse primary HIV-1 strains. The D19 epitope was conserved in the majority (23/29; 79.3%) of the subtype-B strains tested, as well as in selected strains from other genetic subtypes. Strikingly, in CCR5-restricted (R5) isolates, the D19 epitope was invariably cryptic, although it could be exposed by addition of soluble CD4 (sCD4); epitope masking was dependent on the native oligomeric structure of Env, since it was not observed with the corresponding monomeric gp120 molecules. By contrast, in CXCR4-using strains (X4 and R5X4), the epitope was constitutively accessible. In accordance with these results, R5 isolates were resistant to neutralization by MAb D19, becoming sensitive only upon addition of sCD4, whereas CXCR4-using isolates were neutralized regardless of the presence of sCD4. Other V3 epitopes examined did not display a similar divergence in accessibility based on coreceptor usage phenotype. These results provide the first evidence of a correlation between HIV-1 biological phenotype and neutralization sensitivity, raising the possibility that the in vivo evolution of HIV-1 coreceptor usage may be influenced by the selective pressure of specific host antibodies.
PMCID: PMC1112133  PMID: 15890935
12.  Alteration of V3 loop context within the envelope of human immunodeficiency virus type 1 enhances neutralization. 
Journal of Virology  1994;68(6):3459-3466.
Neutralization of a chimeric human immunodeficiency virus (HIV) type 1, containing the V3 loop of the MN isolate substituted within the HXB2 envelope, was enhanced up to 20-fold compared with the HXB2 or MN parental isolates by human HIV-positive sera. MN V3 loop-specific monoclonal antibodies were better able to recognize the chimeric virus compared with MN, staining a greater percentage of infected cells and exhibiting slight increases in relative affinity with a concomitant increase in neutralization titer. Competition analysis revealed that enhanced neutralization by human HIV-positive sera of the chimera was attributable in some cases to better reactivity with the linear V3 loop epitope but in others to conformational loop epitopes or previously cryptic or poorly recognized epitopes outside the loop region. Mice primed with a vaccinia virus-chimeric envelope recombinant and boosted with gp160 developed a spectrum of antibodies different from that of mice similarly immunized with HXB2 or MN recombinants or that of naturally infected humans. The chimeric envelope elicited antibodies with enhanced binding to the native MN V3 loop; however, the sites seen by the BALB/c mice were not neutralizing epitopes. Nevertheless, similar to the observations made with use of human sera, the chimeric virus was more readily neutralized by all of the immune mouse sera, an effect apparently mediated by non-V3 loop epitopes. These studies illustrate that not only the V3 loop sequence and conformation but also its context within the viral envelope influence neutralization.
PMCID: PMC236848  PMID: 7514675
13.  Limited Neutralizing Antibody Specificities Drive Neutralization Escape in Early HIV-1 Subtype C Infection 
PLoS Pathogens  2009;5(9):e1000598.
We previously showed that HIV-1 subtype C viruses elicit potent but highly type-specific neutralizing antibodies (nAb) within the first year of infection. In order to determine the specificity and evolution of these autologous nAbs, we examined neutralization escape in four individuals whose responses against the earliest envelope differed in magnitude and potency. Neutralization escape occurred in all participants, with later viruses showing decreased sensitivity to contemporaneous sera, although they retained sensitivity to new nAb responses. Early nAb responses were very restricted, occurring sequentially and targeting only two regions of the envelope. In V1V2, limited amino acid changes often involving indels or glycans, mediated partial or complete escape, with nAbs targeting the V1V2 region directly in 2 cases. The alpha-2 helix of C3 was also a nAb target, with neutralization escape associated with changes to positively charged residues. In one individual, relatively high titers of anti-C3 nAbs were required to drive genetic escape, taking up to 7 weeks for the resistant variant to predominate. Thereafter titers waned but were still measurable. Development of this single anti-C3 nAb specificity was associated with a 7-fold drop in HIV-1 viral load and a 4-fold rebound as the escape mutation emerged. Overall, our data suggest the development of a very limited number of neutralizing antibody specificities during the early stages of HIV-1 subtype C infection, with temporal fluctuations in specificities as escape occurs. While the mechanism of neutralization escape appears to vary between individuals, the involvement of limited regions suggests there might be common vulnerabilities in the HIV-1 subtype C transmitted envelope.
Author Summary
Most HIV-1 infected individuals develop neutralizing antibodies against their own virus, termed an autologous neutralizing response. It is known that this response exerts pressure on the envelope of HIV, the target of such antibodies, resulting in neutralization escape. Here we have identified the targets of these antibodies and the precise genetic basis of neutralization escape in 4 individuals infected with HIV-1 subtype C. We show that V1V2 is commonly involved in escape, and that the C3 region is also a target in some cases. The latter observation confirms this region is exposed in subtype C, unlike subtype B. We show that neutralization escape is conferred by a few amino acid mutations, some of which are outside the antibody target site. Moreover, escape from these limited specificities even within a single individual occurs via a variety of different pathways involving substitutions, indels and glycan shifts. The finding in 2 individuals that an anti-C3 response developed first, followed by an anti-V1V2 response, suggests there may be specific regions of envelope particularly vulnerable to antibody neutralization. Overall, we propose a mechanistic explanation for how HIV-1 epitopes drive sequential waves of neutralization escape in early subtype C infection.
PMCID: PMC2742164  PMID: 19763271
14.  Crystal Structures of Human Immunodeficiency Virus Type 1 (HIV-1) Neutralizing Antibody 2219 in Complex with Three Different V3 Peptides Reveal a New Binding Mode for HIV-1 Cross-Reactivity 
Journal of Virology  2006;80(12):6093-6105.
Human monoclonal antibody 2219 is a neutralizing antibody isolated from a human immunodeficiency virus type 1-infected individual. 2219 was originally selected for binding to a V3 fusion protein and can neutralize primary isolates from subtypes B, A, and F. Thus, 2219 represents a cross-reactive, human anti-V3 antibody. Fab 2219 binds to one face of the variable V3 β-hairpin, primarily contacting conserved residues on the N-terminal β-strand of V3, leaving the V3 crown or tip largely accessible. Three V3/2219 complexes reveal the antibody-bound conformations for both the N- and C-terminal regions that flank the V3 crown and illustrate how twisting of the V3 loop alters the relative dispositions and pairing of the amino acids in the adjacent V3 β-strands and how the antibody can accommodate V3 loops with different sequences.
PMCID: PMC1472588  PMID: 16731948
15.  Cross-neutralization of human immunodeficiency virus type 1 and 2 and simian immunodeficiency virus isolates. 
Journal of Virology  1992;66(6):3602-3608.
In contrast to infrequent and low-titer cross-neutralization of human immunodeficiency virus type 1 (HIV-1) isolates by HIV-2- and simian immunodeficiency virus (SIV)-positive sera, extensive cross-neutralization of HIV-2NIH-Z, SIVMAC251, and SIVAGM208K occurs with high titer, suggesting conservation of epitopes and mechanism(s) of neutralization. The V3 regions of HIV-2 and SIV isolates, minimally related to the HIV-1 homolog, share significant sequence homology and are immunogenic in monkeys as well as in humans. Whereas the crown of the V3 loop is cross-reactive among HIV-1 isolates and elicits neutralizing antibodies of broad specificity, the SIV and especially HIV-2 crown peptides were not well recognized by cross-neutralizing antisera. V3 loop peptides of HIV-2 isolates did not elicit neutralizing antibodies in mice, guinea pigs, or a goat and together with SIV V3 peptides did not inhibit serum neutralization of HIV-2 and SIV. Thus, the V3 loops of HIV-2 and SIV do not appear to constitute simple linear neutralizing epitopes. In view of the immunogenicity of V3 peptides, the failure of conserved crown peptides to react with natural sera implies a significant role of loop conformation in antibody recognition. Our studies suggest that in addition to their grouping by envelope genetic relatedness, HIV-2 and SIV are neutralized similarly to each other but differently from HIV-1. The use of linear peptides of HIV-2 and SIV as immunogens may require greater attention to microconformation, and alternate subunit approaches may be needed in exploiting these viruses as vaccine models. Such approaches may also be applicable to the HIV-1 system in which conformational epitopes, in addition to the V3 loop, participate in virus neutralization.
PMCID: PMC241142  PMID: 1374810
16.  Proline-rich tandem repeats of antibody complementarity-determining regions bind and neutralize human immunodeficiency virus type 1 particles. 
Journal of Virology  1996;70(10):6557-6562.
The proline-rich tandem repeat domain of human mucin MUC1 forms an extended structure containing large repeating loops that are crested by a turn. We show that the repeating-loop structure of MUC1 can be replaced by an antibody complementarity-determining region loop of a human immunodeficiency virus type 1 (HIV-1)-specific neutralizing antibody to create a chimeric, multivalent, mucin-like, anti-HIV-1 compound. We used 8 residues of an antibody molecule to replace 8 of 20 residues of the MUC1 tandem-repeat sequence. The antiviral peptide discussed here contains three copies of a 20-residue tandem repeat, (IYYDYEEDPAPGSTAPPAHG)3, for a total of 60 residues. We demonstrate that the mucin-antibody chimera retains the binding specificity of the parent antibody (monoclonal antibody F58), GPGR of the HIV-1 gp120 V3 neutralizing epitope, and the ability to neutralize virus particles. In inhibition enzyme-linked immunosorbent assay, the mucin-antibody chimeric peptide could inhibit 71 to 84% of binding to a V3 loop peptide by monoclonal antibodies known to be specific for GPGR in the V3 loop. The mucin-antibody chimeric peptide could also inhibit monoclonal antibody binding to native gp120 captured from virus particles. In addition, the chimeric peptide neutralized the homologous HIV-IIIB virus in a standard neutralization assay. The methods of antiviral peptide design and construction presented here are general and theoretically limited only by the size of the antibody repertoire. This approach could be used to synthesize peptides for a variety of therapeutic applications.
PMCID: PMC190696  PMID: 8794290
17.  Insensitivity of Paediatric HIV-1 Subtype C Viruses to Broadly Neutralising Monoclonal Antibodies Raised against Subtype B 
PLoS Medicine  2006;3(7):e255.
A Phase I clinical trial has been proposed that uses neutralising monoclonal antibodies (MAbs) as passive immunoprophylaxis to prevent mother-to-child transmission of HIV-1 in South Africa. To assess the suitability of such an approach, we determined the sensitivity of paediatric HIV-1 subtype C viruses to the broadly neutralising MAbs IgG1b12, 2G12, 2F5, and 4E10.
Methods and Findings
The gp160 envelope genes from seven children with HIV-1 subtype C infection were cloned and used to construct Env-pseudotyped viruses that were tested in a single-cycle neutralisation assay. The epitopes defining three of these MAbs were determined from sequence analysis of the envelope genes. None of the seven HIV-1 subtype C pseudovirions was sensitive to 2G12 or 2F5, which correlated with the absence of crucial N-linked glycans that define the 2G12 epitope and substitutions of residues integral to the 2F5 epitope. Four viruses were sensitive to IgG1b12, and all seven viruses were sensitive to 4E10.
Only 4E10 showed significant activity against HIV-1 subtype C isolates, while 2G12 and 2F5 MAbs were ineffective and IgG1b12 was partly effective. It is therefore recommended that 2G12 and 2F5 MAbs not be used for passive immunization experiments in southern Africa and other regions where HIV-1 subtype C viruses predominate.
Editors' Summary
AIDS is caused by HIV. By killing the cells of the body's immune system, HIV infection makes people vulnerable to many potentially fatal bacterial and viral diseases. HIV is most commonly spread through unprotected sex with an infected partner but it can also pass from mother to child during late pregnancy or birth, or through breast milk. At least one in four infected women will transmit HIV to their babies if left untreated. But if infected women are treated with drugs that fight HIV—so-called antiretrovirals—during late pregnancy and if breastfeeding does not occur, only one to two babies in 100 will become infected with HIV. In addition, elective Caesarian section has been found to be protective against HIV infection. Implementation of this approach has greatly reduced mother-to-child transmission in developed countries, but most HIV-infected women live in developing countries where access to antiretrovirals is limited. In these cases, treatment of pregnant women (during pregnancy and delivery) and their newborn babies with a single dose of one antiretroviral drug, which can halve HIV transmission, is used, even though WHO/UNAIDS recommends simple antenatal, intrapartum, and postnatal antiretroviral regimens to achieve levels of less than 5% transmission in resource poor settings. These strategies will not have an impact on breastmilk transmission, which accounts for half the transmissions in these settings.
Why Was This Study Done?
One way to reduce breastmilk transmission of HIV might be by “passive immunization.” In this, newborn babies would be injected with HIV-specific antibodies—proteins that stick to molecules on the surface of HIV. Because the virus uses these molecules to invade the baby's immune cells, injected antibodies might stop HIV from the mother becoming established in her offspring. Four antibodies have been made in the laboratory—so-called human monoclonal antibodies—that bind to the surface of HIV subtype B, which is found mainly in Europe and North America, and stop HIV from killing human cells. However, most HIV isolated in Africa is subtype C, so in this study researchers have tested whether these antibodies prevent HIV subtype C killing cells grown in the laboratory. It is important, they argue, that antibodies should be shown to work outside the body before testing passive immunization in babies.
What Did the Researchers Do and Find?
The researchers isolated several subtype C viruses from babies born in Johannesburg, South Africa, and made artificial viruses (known as “pseudotyped” viruses) from them. These artificial viruses could then be used in tests to see whether the human monoclonal antibodies could prevent the viruses infecting human cells in a laboratory test, that is, whether the viruses were “sensitive” to the antibodies. All the viruses were insensitive to two of the antibodies (2G12 and 2F5), and the researchers show that this was because the viruses lacked the specific parts of the HIV surface molecules recognized by these antibodies. Four of the viruses were sensitive to an antibody called IgG1b12, and all were sensitive to antibody 4E10, albeit at high concentrations that might be difficult to achieve in people. Finally, the researchers report that the sensitivity of the viruses was not enhanced by using all four antibodies at the same time.
What Do These Findings Mean?
Given these results, the researchers warn against using 2G12 and 2F5 antibodies for passive immunization to prevent mother-to-child transmission, in particular postnatal transmission, in areas where most people are infected with HIV subtype C viruses. Furthermore, because animal studies have indicated that only combinations of at least three monoclonal antibodies with activity against HIV in laboratory tests provide complete protection against HIV infection, the researchers question whether any clinical trials on passive immunization should be started with currently available antibodies. Their doubts about such trials are heightened by observations that 4E10 and 2F5 react against antigens present on human cells, which might make them unsafe for use in people, although so far no adverse effects have been seen in adults treated with these antibodies. However, these experiments used an artificial laboratory-based assay and it's possible that these antibodies might kill HIV subtype C more effectively in people; other components of the immune system might help them deal with the virus. If clinical studies of these antibodies do go ahead, it is essential that the babies in these trials must be carefully monitored to ensure that the antibodies are safe, and they and their mothers should also be given access to optimal antiretroviral prophylaxis according to WHO/UNAIDS guidelines. In a related PLoS Medicine Perspective paper (, Miroslaw Gorny1 and Susan Zolla-Pazner discuss the study further and stress the critical need to determine if passive immunization with such antibodies could decrease mother-to-child transmission of HIV, and if so what the best antibodies would be.
Additional Information.
Please access these Web sites via the online version of this summary at
•  National Institute of Allergy and Infectious Diseases fact sheets on HIV infection and AIDS
•  US Department of Health and Human Services information on HIV/AIDS, including clinical guidelines and fact sheets on preventing transmission from mother to child
•  US Centers for Disease Control and Prevention information on HIV/AIDS, including pages on the prevention of mother-to-child transmission
•  MedlinePlus encyclopedia entry on HIV/AIDS
•  Preventing mother-to-child transmission of HIV Web page
Assessment of viruses from seven children with HIV-1 subtype C infection showed generally poor sensitivity to four monoclonal antibodies proposed for a trial of passive immunoprophylaxis to prevent mother-to-child transmission.
PMCID: PMC1502151  PMID: 16834457
18.  Regional Clustering of Shared Neutralization Determinants on Primary Isolates of Clade C Human Immunodeficiency Virus Type 1 from South Africa 
Journal of Virology  2002;76(5):2233-2244.
Clade C is one of the most prevalent genetic subtypes of human immunodeficiency virus type 1 (HIV-1) in the world today and one of the least studied with respect to neutralizing antibodies. Most information on HIV-1 serology as it relates to neutralization is derived from clade B. Clade C primary isolates of HIV-1 from South Africa and Malawi were shown here to resemble clade B isolates in their resistance to inhibition by soluble CD4 and their sensitivity to neutralization by human monoclonal antibody immunoglobulin G1b12 and, to a lesser extent, 2F5. Unlike clade B isolates, however, all 16 clade C isolates examined resisted neutralization by 2G12. Infection with clade C HIV-1 in a cohort of female sex workers in South Africa generated antibodies that neutralized the autologous clade C isolate and T-cell-line-adapted (TCLA) strains of clade B. Neutralization of clade B TCLA strains was much more sensitive to the presence of autologous gp120 V3 loop peptides compared to the neutralization of clade C isolates in most cases. Thus, the native structure of gp120 on primary isolates of clade C will likely pose a challenge for neutralizing antibody induction by candidate HIV-1 vaccines much the same as it has for clade B. The autologous neutralizing antibody response following primary infection with clade C HIV-1 in South Africa matured slowly, requiring at least 4 to 5 months to become detectable. Once detectable, extensive cross-neutralization of heterologous clade C isolates from South Africa was observed, suggesting an unusual degree of shared neutralization determinants at a regional level. This high frequency of cross-neutralization differed significantly from the ability of South African clade C serum samples to neutralize clade B isolates but did not differ significantly from results of other combinations of clade B and C reagents tested in checkerboard assays. Notably, two clade C serum samples obtained after less than 2 years of infection neutralized a broad spectrum of clade B and C isolates. Other individual serum samples showed a significant clade preference in their neutralizing activity. Our results suggest that clades B and C are each comprised of multiple neutralization serotypes, some of which are more clade specific than others. The clustering of shared neutralization determinants on clade C primary HIV-1 isolates from South Africa suggests that neutralizing antibodies induced by vaccines will have less epitope diversity to overcome at a regional level.
PMCID: PMC135941  PMID: 11836401
19.  The Thai Phase III HIV Type 1 Vaccine Trial (RV144) Regimen Induces Antibodies That Target Conserved Regions Within the V2 Loop of gp120 
AIDS Research and Human Retroviruses  2012;28(11):1444-1457.
The Thai Phase III clinical trial (RV144) showed modest efficacy in preventing HIV-1 acquisition. Plasma collected from HIV-1-uninfected trial participants completing all injections with ALVAC-HIV (vCP1521) prime and AIDSVAX B/E boost were tested for antibody responses against HIV-1 gp120 envelope (Env). Peptide microarray analysis from six HIV-1 subtypes and group M consensus showed that vaccination induced antibody responses to the second variable (V2) loop of gp120 of multiple subtypes. We further evaluated V2 responses by ELISA and surface plasmon resonance using cyclic (Cyc) and linear V2 loop peptides. Thirty-one of 32 vaccine recipients tested (97%) had antibody responses against Cyc V2 at 2 weeks postimmunization with a reciprocal geometric mean titer (GMT) of 1100 (range: 200–3200). The frequency of detecting plasma V2 antibodies declined to 19% at 28 weeks post-last injection (GMT: 110, range: 100–200). Antibody responses targeted the mid-region of the V2 loop that contains conserved epitopes and has the amino acid sequence KQKVHALFYKLDIVPI (HXB2 Numbering sequence 169–184). Valine at position 172 was critical for antibody binding. The frequency of V3 responses at 2 weeks postimmunization was modest (18/32, 56%) with a GMT of 185 (range: 100–800). In contrast, naturally infected HIV-1 individuals had a lower frequency of antibody responses to V2 (10/20, 50%; p=0.003) and a higher frequency of responses to V3 (19/20, 95%), with GMTs of 400 (range: 100–3200) and 3570 (range: 200–12,800), respectively. RV144 vaccination induced antibodies that targeted a region of the V2 loop that contains conserved epitopes. Early HIV-1 transmission events involve V2 loop interactions, raising the possibility that anti-V2 antibodies in RV144 may have contributed to viral inhibition.
PMCID: PMC3484815  PMID: 23035746
20.  Antibody Responses Elicited in Macaques Immunized with Human Immunodeficiency Virus Type 1 (HIV-1) SF162-Derived gp140 Envelope Immunogens: Comparison with Those Elicited during Homologous Simian/Human Immunodeficiency Virus SHIVSF162P4 and Heterologous HIV-1 Infection†  
Journal of Virology  2006;80(17):8745-8762.
The antibody responses elicited in rhesus macaques immunized with soluble human immunodeficiency virus (HIV) Env gp140 proteins derived from the R5-tropic HIV-1 SF162 virus were analyzed and compared to the broadly reactive neutralizing antibody responses elicited during chronic infection of a macaque with a simian/human immunodeficiency virus (SHIV) expressing the HIV-1 SF162 Env, SHIVSF162P4, and humans infected with heterologous HIV-1 isolates. Four gp140 immunogens were evaluated: SF162gp140, ΔV2gp140 (lacking the crown of the V2 loop), ΔV3gp140 (lacking the crown of the V3 loop), and ΔV2ΔV3gp140 (lacking both the V2 and V3 loop crowns). SF162gp140 and ΔV2gp140 have been previously evaluated by our group in a pilot study, but here, a more comprehensive analysis of their immunogenic properties was performed. All four gp140 immunogens elicited stronger anti-gp120 than anti-gp41 antibodies and potent homologous neutralizing antibodies (NAbs) that primarily targeted the first hypervariable region (V1 loop) of gp120, although SF162gp140 also elicited anti-V3 NAbs. Heterologous NAbs were elicited by SF162gp140 and ΔV2gp140 but were weak in potency and narrow in specificity. No heterologous NAbs were elicited by ΔV3gp140 or ΔV2ΔV3gp140. In contrast, the SHIVSF162P4-infected macaque and HIV-infected humans generated similar titers of anti-gp120 and anti-gp41 antibodies and NAbs of significant breadth against primary HIV-1 isolates, which did not target the V1 loop. The difference in V1 loop immunogenicity between soluble gp140 and virion-associated gp160 Env proteins derived from SF162 may be the basis for the observed difference in the breadth of neutralization in sera from the immunized and infected animals studied here.
PMCID: PMC1563892  PMID: 16912322
21.  Analysis of Genetic Variability within the Immunodominant Epitopes of Envelope gp41 from Human Immunodeficiency Virus Type 1 (HIV-1) Group M and Its Impact on HIV-1 Antibody Detection 
Journal of Clinical Microbiology  2000;38(2):773-780.
The serodiagnosis of human immunodeficiency virus type 1 (HIV-1) infection primarily relies on the detection of antibodies, most of which are directed against the immunodominant regions (IDR) of HIV-1 structural proteins. Among these, the N-terminal region of gp41 contains cluster I (amino acids [aa] 580 to 623), comprising the cytotoxic T-lymphocyte epitope (AVERYLKDQQLL) and the cysteine loop (CSGKLIC), and cluster II (aa 646 to 682), comprising an ectodomain region (ELDKWA). To delineate the epitope diversity within clusters I and II and to determine whether the diversity affects serologic detection by U.S. Food and Drug Administration (FDA)-licensed enzyme immunoassay (EIA) kits, gp41 Env sequences from 247 seropositive persons infected with HIV-1 group M, subtypes A (n = 42), B (n = 62), B′ (n = 13), C (n = 38), D (n = 41), E (n = 18), F (n = 27), and G (n = 6), and 6 HIV-1-infected but persistently seronegative (HIPS) persons were analyzed. While all IDR were highly conserved among both seropositive and HIPS persons, minor amino acid substitutions (<20% for any one residue, mostly conservative) were observed for all subtypes, except for B′, in comparison with the consensus sequence for each subtype. Most importantly, none of the observed substitutions among the group M plasma specimens affected antibody detection, since all specimens (n = 152) tested positive with all five FDA-licensed EIA kits. Furthermore, all specimens reacted with a group M consensus gp41 peptide (WGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICTTAVPWNASW), and high degrees of cross-reactivity (>80%) were observed with an HIV-1 group N peptide, an HIV-1 group O peptide, and a peptide derived from the homologous region of gp41 from simian immunodeficiency virus from chimpanzee (SIVcpz). Taken together, these data indicate that the minor substitutions observed within the IDR of gp41 of HIV-1 group M subtypes do not affect antibody recognition and that all HIV-1-seropositive specimens containing the observed substitutions react with the FDA-licensed EIA kits regardless of viral genotype and geographic origin.
PMCID: PMC86201  PMID: 10655384
22.  Structure determination of an anti-HIV-1 Fab 447-52D–peptide complex from an epitaxially twinned data set 
Separation of two individual lattices within an epitaxially twinned data set allowed the crystal structure of the V3-specific neutralizing antibody 447-52D in complex with a V3 peptide (UG1033) to be determined. The structure confirms that the neutralization breadth of Fab 447-52D is likely to be attributable to the extensive focus on main-chain hydrogen-bond interactions with the peptide that permit the recognition of a range of V3 sequences.
Although antibodies against the third variable loop (V3) of the HIV-1 viral envelope glycoprotein are among the first neutralizing antibodies to be detected in infected individuals, they are normally restricted in their specificity. X-ray crystallo­graphic studies of V3-specific antibodies have con­tributed to a more thorough understanding of recognition of this epitope and of conserved features in the V3 loop that could potentially aid in the design of a multi-component vaccine. The human antibody 447-52D exhibits relatively broad neutralization of primary viral isolates compared with other V3-loop antibodies. A crystal structure of Fab 447-52D in complex with a V3 peptide (UG1033) was determined at 2.1 Å resolution. The structure was determined using an epitaxially twinned data set and in-house programs to detect and remove overlapping reflections. Although the processed data have lower than desired completeness and slightly higher than normal R values for the resolution, good-quality electron-density maps were obtained that enabled structure determination. The structure revealed an extended CDR H3 loop that forms a β-sheet with the peptide, with the predominant contacts being main-chain hydrogen bonds. The V3 peptide and Fab show high structural homology with the previously reported structures of other Fab 447-52D complexes, reinforcing the idea that the V3 loop may adopt a small set of conserved structures, particularly around the crown of the β-hairpin.
PMCID: PMC2631122  PMID: 18566514
antibodies; HIV-1; twinning; V3 loop
23.  The C3-V4 Region Is a Major Target of Autologous Neutralizing Antibodies in Human Immunodeficiency Virus Type 1 Subtype C Infection▿  
Journal of Virology  2007;82(4):1860-1869.
The early autologous neutralizing antibody response in human immunodeficiency virus type 1 (HIV-1) subtype C infections is often characterized by high titers, but the response is type specific with little to no cross-neutralizing activity. The specificities of these early neutralizing antibodies are not known; however, the type specificity suggests that they may target the variable regions of the envelope. Here, we show that cross-reactive anti-V3 antibodies developed within 3 to 12 weeks in six individuals but did not mediate autologous neutralization. Using a series of chimeric viruses, we found that antibodies directed at the V1V2, V4, and V5 regions contributed to autologous neutralization in some individuals, with V1V2 playing a more substantial role. However, these antibodies did not account for the total neutralizing capacity of these sera against the early autologous virus. Antibodies directed against the C3-V4 region were involved in autologous neutralization in all four sera studied. In two sera, transfer of the C3-V4 region rendered the chimera as sensitive to antibody neutralization as the parental virus. Although the C3 region, which contains the highly variable α2-helix was not a direct target in most cases, it contributed to the formation of neutralization epitopes as substitution of this region resulted in neutralization resistance. These data suggest that the C3 and V4 regions combine to form important structural motifs and that epitopes in this region are major targets of the early autologous neutralizing response in HIV-1 subtype C infection.
PMCID: PMC2258729  PMID: 18057243
24.  A Novel Human Antibody against Human Immunodeficiency Virus Type 1 gp120 Is V1, V2, and V3 Loop Dependent and Helps Delimit the Epitope of the Broadly Neutralizing Antibody Immunoglobulin G1 b12 
Journal of Virology  2003;77(12):6965-6978.
The V1/V2 and V3 loops are proximal to the CD4 binding site (CD4bs) of human immunodeficiency virus type 1 (HIV-1) gp120 and undergo conformational change upon CD4 receptor engagement by the HIV-1 envelope spike. Nearly all of the reported monoclonal antibodies (MAbs) against the CD4bs exhibit a very limited capacity to neutralize HIV-1. However, one such human MAb, immunoglobulin G1 (IgG1) b12, is uniquely able to neutralize primary isolates across subtypes with considerable potency. The molecular basis for the anti-HIV-1 activity of b12 is not fully understood but is relevant to vaccine design. Here we describe a novel human MAb, 4KG5, whose binding to monomeric gp120 is moderately enhanced by IgG1 b12. In sharp contrast, 4KG5 binding to gp120 is inhibited by soluble CD4 (sCD4) and by all other (n = 14) anti-CD4bs MAbs tested. 4KG5 is unable to recognize gp120 in which either V1, V2, or V3 has been deleted, and MAbs against the V2 or V3 loops inhibit the binding of 4KG5 to gp120. Moreover, 4KG5 is able to inhibit the binding of the CD4-induced MAbs 17b and X5 in the absence of sCD4, whereas 17b and X5 only weakly inhibit the binding of 4KG5 to gp120. Mutagenesis of gp120 provides further evidence of a discontinuous epitope of 4KG5 that is formed by the V1/V2 loop, the V3 loop, and a portion of the bridging sheet (C4). 4KG5 was isolated as a single-chain Fv from a phage display library constructed from the bone marrow of an HIV-1-seropositive subject (FDA2) whose serum neutralizes HIV-1 across subtypes. Despite its source, we observed no significant neutralization with 4KG5 against the autologous (R2) virus and several other strains of HIV-1. The results suggest a model in which antibody access to the CD4bs on the envelope spike of HIV-1 is restricted by the orientation and/or dynamics of the V1/V2 and V3 loops, and b12 avoids these restrictions.
PMCID: PMC156200  PMID: 12768015
25.  Subtype-Specific Conformational Differences within the V3 Region of Subtype B and Subtype C Human Immunodeficiency Virus Type 1 Env Proteins▿  
Journal of Virology  2007;82(2):903-916.
The V3 region of the human immunodeficiency virus type 1 gp120 Env protein is a key domain in Env due to its role in interacting with the coreceptors CCR5 and CXCR4. We examined potential subtype-specific V3 region differences by comparing patterns of amino acid variability and probing for subtype-specific structures using 11 anti-V3 monoclonal antibodies (V3 MAbs). Differences between the subtypes in patterns of variability were most evident in the stem and turn regions of V3 (positions 9 to 24), with the two subtypes being very similar in the base region. The characteristics of the binding of V3 MAbs to Env proteins of the subtype B virus JR-FL and the subtype C virus BR025 suggested three patterns, as each group of MAbs recognized a specific conformation- or sequence-based epitope. Viruses pseudotyped with Env from JR-FL and BR025 were resistant to neutralization by the V3 MAbs, although the replacement of the Env V3 region of the SF162 virus with the JR-FL V3 created a pseudotyped virus that was hypersensitive to neutralization. A single mutation in V3 (H13R) made this chimeric Env selectively resistant to one group of V3 MAbs, consistent with the mAb binding properties. We hypothesize that there are intrinsic differences in V3 conformation between subtype B and subtype C that are localized to the stem and turn regions and that these differences have two important biological consequences: first, subtype B and subtype C V3 regions can have subtype-specific epitopes that will inherently limit antibody cross-reactivity, and second, V3 conformational differences may potentiate the frequent evolution of R5- into X4-tropic variants of subtype B but limit subtype C virus from using the same mechanism to evolve X4-tropic variants as efficiently.
PMCID: PMC2224581  PMID: 18003735

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