The development of an effective AIDS vaccine has been a formidable task, but remains a critical necessity. The well conserved membrane-proximal external region (MPER) of the HIV-1 gp41 glycoprotein is one of the crucial targets for AIDS vaccine development, as it has the necessary attribute of being able to elicit antibodies capable of neutralizing diverse isolates of HIV.
Guided by X-ray crystallography, molecular modeling, combinatorial chemistry, and powerful selection techniques, we designed and produced six combinatorial libraries of chimeric human rhinoviruses (HRV) displaying the MPER epitopes corresponding to mAbs 2F5, 4E10, and/or Z13e1, connected to an immunogenic surface loop of HRV via linkers of varying lengths and sequences. Not all libraries led to viable chimeric viruses with the desired sequences, but the combinatorial approach allowed us to examine large numbers of MPER-displaying chimeras. Among the chimeras were five that elicited antibodies capable of significantly neutralizing HIV-1 pseudoviruses from at least three subtypes, in one case leading to neutralization of 10 pseudoviruses from all six subtypes tested.
Optimization of these chimeras or closely related chimeras could conceivably lead to useful components of an effective AIDS vaccine. While the MPER of HIV may not be immunodominant in natural infection by HIV-1, its presence in a vaccine cocktail could provide critical breadth of protection.
The swarm of quasispecies that evolves in each HIV-1-infected individual represents a source of closely related Env protein variants that can be used to explore various aspects of HIV-1 biology. In this study, we made use of these variants to identify mutations that confer sensitivity and resistance to the broadly neutralizing antibodies found in the sera of selected HIV-1-infected individuals. For these studies, libraries of Env proteins were cloned from infected subjects and screened for infectivity and neutralization sensitivity. The nucleotide sequences of the Env proteins were then compared for pairs of neutralization-sensitive and -resistant viruses. In vitro mutagenesis was used to identify the specific amino acids responsible for the neutralization phenotype. All of the mutations altering neutralization sensitivity/resistance appeared to induce conformational changes that simultaneously enhanced the exposure of two or more epitopes located in different regions of gp160. These mutations appeared to occur at unique positions required to maintain the quaternary structure of the gp160 trimer, as well as conformational masking of epitopes targeted by neutralizing antibodies. Our results show that sequences in gp41, the CD4 binding site, and the V2 domain all have the ability to act as global regulators of neutralization sensitivity. Our results also suggest that neutralization assays designed to support the development of vaccines and therapeutics targeting the HIV-1 Env protein should consider virus variation within individuals as well as virus variation between individuals.
Development of a vaccine for HIV-1 requires a detailed understanding of the neutralizing antibody responses that can be experimentally elicited to difficult-to-neutralize primary isolates. Rabbits were immunized with the gp120 subunit of HIV-1 JR-CSF envelope (Env) using a DNA-prime protein-boost regimen. We analyzed five sera that showed potent autologous neutralizing activity (IC50s at ∼103 to 104 serum dilution) against pseudoviruses containing Env from the primary isolate JR-CSF but not from the related isolate JR-FL. Pseudoviruses were created by exchanging each variable and constant domain of JR-CSF gp120 with that of JR-FL or with mutations in putative N-glycosylation sites. The sera contained different neutralizing activities dependent on C3 and V5, C3 and V4, or V4 regions located on the glycan-rich outer domain of gp120. All sera showed enhanced neutralizing activity toward an Env variant that lacked a glycosylation site in V4. The JR-CSF gp120 epitopes recognized by the sera are generally distinct from those of several well characterized mAbs (targeting conserved sites on Env) or other type-specific responses (targeting V1, V2, or V3 variable regions). The activity of one serum requires specific glycans that are also important for 2G12 neutralization and this serum blocked the binding of 2G12 to gp120. Our findings show that different fine specificities can achieve potent neutralization of HIV-1, yet this strong activity does not result in improved breadth.
Recently, several broadly neutralizing monoclonal antibodies (bnMAbs) directed to the CD4-binding site (CD4bs) of gp120 have been isolated from HIV-1-positive donors. These include VRC01, 3BNC117, and NIH45-46, all of which are capable of neutralizing about 90% of circulating HIV-1 isolates and all of which induce conformational changes in the HIV-1 gp120 monomer similar to those induced by the CD4 receptor. In this study, we characterize PGV04 (also known as VRC-PG04), a MAb with potency and breadth that rivals those of the prototypic VRC01 and 3BNC117. When screened on a large panel of viruses, the neutralizing profile of PGV04 was distinct from those of CD4, b12, and VRC01. Furthermore, the ability of PGV04 to neutralize pseudovirus containing single alanine substitutions exhibited a pattern distinct from those of the other CD4bs MAbs. In particular, substitutions D279A, I420A, and I423A were found to abrogate PGV04 neutralization. In contrast to VRC01, PGV04 did not enhance the binding of 17b or X5 to their epitopes (the CD4-induced [CD4i] site) in the coreceptor region on the gp120 monomer. Furthermore, in contrast to CD4, none of the anti-CD4bs MAbs induced the expression of the 17b epitope on cell surface-expressed cleaved Env trimers. We conclude that potent CD4bs bnMAbs can display differences in the way they recognize and access the CD4bs and that mimicry of CD4, as assessed by inducing conformational changes in monomeric gp120 that lead to enhanced exposure of the CD4i site, is not uniquely correlated with effective neutralization at the site of CD4 binding on HIV-1.
We previously established that at 3 years postseroconversion, ∼30% of HIV-infected individuals have cross-reactive neutralizing activity (CrNA) in their sera. Here we studied the kinetics with which CrNA develops and how these relate to the development of autologous neutralizing activity as well as viral escape and diversification. For this purpose, sera from five individuals with CrNA and one elite neutralizer that were obtained at three monthly intervals in the first year after seroconversion and at multiple intervals over the disease course were tested for neutralizing activity against an established multiclade panel of six viruses. The same serum samples, as well as sera from three individuals who lacked CrNA, were tested for their neutralizing activities against autologous clonal HIV-1 variants from multiple time points covering the disease course from seroconversion onward. The elite neutralizer already had CrNA at 9.8 months postseroconversion, in contrast with the findings for the other five patients, in whom CrNA was first detected at 20 to 35 months postseroconversion and peaked around 35 months postseroconversion. In all patients, CrNA coincided with neutralizing activity against autologous viruses that were isolated <12 months postseroconversion, while viruses from later time points had already escaped autologous neutralizing activity. Also, the peak in gp160 sequence diversity coincided with the peak of CrNA titers. Individuals who lacked CrNA had lower peak autologous neutralizing titers, viral escape, and sequence diversity than individuals with CrNA. A better understanding of the underlying factors that determine the presence of CrNA or even an elite neutralizer phenotype may aid in the design of an HIV-1 vaccine.
Broadly neutralizing antibodies (bnAbs) against highly variable viral pathogens are much sought-after to treat or protect against global circulating viruses. We have probed the neutralizing antibody repertoires of four HIV-infected donors with remarkably broad and potent neutralizing responses and rescued 17 new monoclonal antibodies (MAbs) that neutralize broadly across clades. Many of the new MAbs are almost 10-fold more potent than the recently described PG9, PG16, and VRC01 bnMAbs and 100-fold more potent than the original prototype HIV bnMAbs1–3. The MAbs largely recapitulate the neutralization breadth found in the corresponding donor serum and many recognize novel epitopes on envelope (Env) glycoprotein gp120, illuminating new targets for vaccine design. Analysis of neutralization by the full complement of anti-HIV bnMAbs now available reveals that certain combinations of antibodies provide significantly more favorable coverage of the enormous diversity of global circulating viruses than others and these combinations might be sought in active or passive immunization regimes. Overall, the isolation of multiple HIV bnMAbs, from several donors, that, in aggregate, provide broad coverage at low concentrations is a highly positive indicator for the eventual design of an effective antibody-based HIV vaccine.
Neutralizing antibodies provide markers for vaccine-induced protective immunity in many viral infections. By analogy, HIV-1 neutralizing antibodies induced by immunization may well predict vaccine effectiveness. Assessment of neutralizing antibodies is therefore of primary importance, but is hampered by the fact that we do not know which assay(s) can provide measures of protective immunity. An international collaboration (NeutNet) involving 18 different laboratories previously compared different assays using monoclonal antibodies (mAbs) and soluble CD4 (Phase I study).
In the present study (Phase II), polyclonal reagents were evaluated by 13 laboratories. Each laboratory evaluated nine plasmas against an 8 virus panel representing different genetic subtypes and phenotypes. TriMab, a mixture of three mAbs, was used as a positive control allowing comparison of the results with Phase I in a total of nine different assays. The assays used either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) (Virus Infectivity Assays, VIA), or Env (gp160)-pseudotyped viruses (pseudoviruses, PSV) produced in HEK293T cells from molecular clones or from uncloned virus. Target cells included PBMC and genetically engineered cell lines in either single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs including extra- or intra-cellular p24 antigen detection, luciferase, beta-galactosidase or green fluorescent protein (GFP) reporter gene expression.
Using TriMab, results of Phase I and Phase II were generally in agreement for six of the eight viruses tested and confirmed that the PSV assay is more sensitive than PBMC (p = 0.014). Comparisons with the polyclonal reagents showed that sensitivities were dependent on both virus and plasma.
Here we further demonstrate clear differences in assay sensitivities that were dependent on both the neutralizing reagent and the virus. Consistent with the Phase I study, we recommend parallel use of PSV and VIA for vaccine evaluation.
Broadly neutralizing antibodies (bNAbs), which develop over time in some HIV-1 infected individuals, define critical epitopes for HIV vaccine design. Using a systematic approach, we have examined neutralization breadth in the sera of about 1,800 HIV-1 infected individuals, primarily infected with non-clade B viruses, and selected donors for monoclonal antibody (mAb) generation. We then used a high-throughput neutralization screen of antibody-containing culture supernatants from approximately 30,000 activated memory B cells from a clade A-infected African donor to isolate two potent mAbs that target a broadly neutralizing epitope. The previously undescribed epitope is preferentially expressed on trimeric Envelope protein and spans conserved regions of variable loops of the gp120 subunit. The results provide a framework for the design of new vaccine candidates for the elicitation of bNAb responses.
A candidate vaccine consisting of human immunodeficiency virus type 1 (HIV-1) subunit gp120 protein (AIDSVAX™ B/B) was found previously to be non-protective despite strong antibody responses against the vaccine antigens. We assessed the magnitude and breadth of neutralizing antibody responses in this trial.
Neutralizing antibodies were measured against highly sensitive (tier 1) and moderately sensitive (tier 2) strains of HIV-1 subtype B in two independent assays. Vaccine recipients were stratified by gender, race and high versus low behavioral risk of HIV-1 acquisition.
Most vaccine recipients mounted potent neutralizing antibody responses against HIV-1MN and a subset of other tier 1 viruses. Occasional weak neutralizing activity was detected against tier 2 viruses. The response against tier 1 and tier 2 viruses was significantly stronger in women than in men. Race and behavioral risk of HIV-1 acquisition had no significant effect on the response. Prior vaccination had little effect on the neutralizing antibody response that arose post infection.
Weak overall neutralizing antibody responses against tier 2 viruses is consistent with a lack of protection in this trial. The magnitude and breadth of neutralization reported here should be useful for identifying improved vaccines.
HIV; vaccines; antibodies
Creation of an effective vaccine for HIV has been an elusive goal of the scientific community for almost 30 years. Neutralizing antibodies are assumed to be pivotal to the success of a prophylactic vaccine but previous attempts to make an immunogen capable of generating neutralizing antibodies to primary “street strain” isolates have resulted in responses of very limited breadth and potency. The objective of the study was to determine the breadth and strength of neutralizing antibodies against autologous and heterologous primary isolates in a cohort of HIV-1 infected Nigerians and to characterize envelopes from subjects with particularly broad or strong immune responses for possible use as vaccine candidates in regions predominated by HIV-1 CRF02_AG and G subtypes. Envelope vectors from a panel of primary Nigerian isolates were constructed and tested with plasma/sera from the same cohort using the PhenoSense HIV neutralizing antibody assay (Monogram Biosciences Inc, USA) to assess the breadth and potency of neutralizing antibodies. The immediate goal of this study was realized by the recognition of three broadly cross-neutralizing sera: (NG2-clade CRF02_AG, NG3-clade CRF02_AG and NG9- clade G). Based on these findings, envelope gp140 sequences from NG2 and NG9, complemented with a gag sequence (Clade G) and consensus tat (CRF02_AG and G) antigens have been codon-optimized, synthesized, cloned and evaluated in BALB/c mice. The intramuscular administration of these plasmid DNA constructs, followed by two booster DNA immunizations, induced substantial specific humoral response against all constructs and strong cellular responses against the gag and tat constructs. These preclinical findings provide a framework for the design of candidate vaccine for use in regions where the HIV-1 epidemic is driven by clades CRF02_AG and G.
A prophylactic vaccine is needed to slow the spread of HIV-1 infection. Optimization of the wild-type envelope glycoproteins to create immunogens that can elicit effective neutralizing antibodies is a high priority. Starting with ten genes encoding subtype B HIV-1 gp120 envelope glycoproteins and using in vitro homologous DNA recombination, we created chimeric gp120 variants that were screened for their ability to bind neutralizing monoclonal antibodies. Hundreds of variants were identified with novel antigenic phenotypes that exhibit considerable sequence diversity. Immunization of rabbits with these gp120 variants demonstrated that the majority can induce neutralizing antibodies to HIV-1. One novel variant, called ST-008, induced significantly improved neutralizing antibody responses when assayed against a large panel of primary HIV-1 isolates. Further study of various deletion constructs of ST-008 showed that the enhanced immunogenicity results from a combination of effective DNA priming, an enhanced V3-based response, and an improved response to the constant backbone sequences.
Understanding the determinants of neutralization sensitivity and resistance is important for the development of an effective human immunodeficiency virus type 1 (HIV-1) vaccine. In these studies, we have made use of the swarm of closely related envelope protein variants (quasispecies) from an extremely neutralization-resistant clinical isolate in order to identify mutations that conferred neutralization sensitivity to antibodies in sera from HIV-1-infected individuals. Here, we describe a virus with a rare mutation at position 179 in the V2 domain of gp120, where replacement of aspartic acid (D) by asparagine (N) converts a virus that is highly resistant to neutralization by multiple polyclonal and monoclonal antibodies, as well as antiviral entry inhibitors, to one that is sensitive to neutralization. Although the V2 domain sequence is highly variable, D at position 179 is highly conserved in HIV-1 and simian immunodeficiency virus (SIV) and is located within the LDI/V recognition motif of the recently described α4β7 receptor binding site. Our results suggest that the D179N mutation induces a conformational change that exposes epitopes in both the gp120 and the gp41 portions of the envelope protein, such as the CD4 binding site and the MPER, that are normally concealed by conformational masking. Our results suggest that D179 plays a central role in maintaining the conformation and infectivity of HIV-1 as well as mediating binding to α4β7.
We analyzed immune responses in chronically HIV-infected individuals who took part in a treatment interruption (TI) trial designed for patients who initiated anti-retroviral therapy within 6 months of seroconversion. In the two subjects that exhibited the best viral control, we detected CD8+ T cell responses against 1-2 Gag epitopes during the early weeks of TI and a subsequent increase in the number of epitopes recognized by the later time points. Each of these subjects developed mutations within the epitopes targeted by the highest magnitude responses. In the subject with the worst viral control, we detected responses against two Gag epitopes throughout the entire TI and no Gag mutations. The magnitude of these responses increased dramatically with time, greatly exceeding those detected in the virologic controllers. The highest levels of contemporaneous autologous neutralizing antibody activity were detected in the virologic controllers, and a subsequent escape mutation developed within the envelope gene of one controller that abrogated the response. These data suggest that immune escape mutations are a sign of viral control during TI, and that the absence of immune escape mutations in the presence of high-levels of viral replication indicates the lack of an effective host immune response.
It is assumed that an effective human immunodeficiency virus type 1 (HIV-1) vaccine should be capable of eliciting neutralizing antibodies. However, even the best antibodies known to date lack neutralizing ability against a significant proportion of primary HIV-1 variants and, despite great efforts, still no immunogen is available that can elicit humoral immunity which is protective against infection or disease progression. We tested sera from 35 participants in the Amsterdam Cohort Studies on HIV-1 infection, who were all infected with HIV-1 subtype B and therapy-naïve at the time of sampling, for neutralizing activity against a panel of 23 tier 2–3 HIV-1 variants, with a minimum of five HIV-1 variants per subtype (A, B, C and D). Strong cross-clade neutralizing activity was detected in sera from seven individuals. Strikingly, sera from 22 of 35 individuals (63 %) neutralized three or more of the six tier 2–3 HIV-1 subtype B viruses in the panel. There was a strong correlation between neutralization titre and breadth in serum. Indeed, the IC50 of sera with strong cross-clade neutralizing activity was significantly higher than the IC50 of sera with cross-subtype B activity, which, in turn, had a higher IC50 than sera with the lowest neutralization breadth. These results imply that humoral immunity, at least in HIV-1 subtype B-infected individuals, is often subtype-specific rather than strain-specific and that the breadth of neutralization is correlated with the titre of neutralizing activity in serum. Considering the difficulties in designing a vaccine that is capable of eliciting cross-clade neutralizing activity, subtype-specific vaccines may be explored as an interesting alternative.
The external domains of the HIV-1 envelope glycoprotein (gp120 and the gp41 ectodomain, collectively known as gp140) contain all known viral neutralization epitopes. Various strategies have been used to create soluble trimers of the envelope to mimic the structure of the native viral protein, including mutation of the gp120-gp41 cleavage site, introduction of disulfide bonds, and fusion to heterologous trimerization motifs. We compared the effects on quaternary structure, antigenicity, and immunogenicity of three such motifs: T4 fibritin, a GCN4 variant, and the E. coli aspartate transcarbamoylase catalytic subunit. Fusion of each motif to the C-terminus of a non-cleavable JRCSF gp140(-) envelope protein led to enhanced trimerization but had limited effects on the antigenic profile and CD4 binding ability of the trimers. Immunization of rabbits provided no evidence that the trimerized gp140(-) constructs induced significantly improved neutralizing antibodies to several HIV-1 pseudoviruses, compared to gp140 lacking a trimerization motif. However, modest differences in both binding specificity and neutralizing antibody responses were observed among the various immunogens.
Vaccine; HIV-1; Envelope; trimerization motifs; ATCase; T4 fibritin; GCN; CD4; monoclonal antibody; immunization; neutralizing antibody
A substantial proportion of human immunodeficiency virus type 1 (HIV-1)-infected individuals has cross-reactive neutralizing activity in serum, with a similar prevalence in progressors and long-term nonprogressors (LTNP). We studied whether disease progression in the face of cross-reactive neutralizing serum activity is due to fading neutralizing humoral immunity over time or to viral escape. In three LTNP and three progressors, high-titer cross-reactive HIV-1-specific neutralizing activity in serum against a multiclade pseudovirus panel was preserved during the entire clinical course of infection, even after AIDS diagnosis in progressors. However, while early HIV-1 variants from all six individuals could be neutralized by autologous serum, the autologous neutralizing activity declined during chronic infection. This could be attributed to viral escape and the apparent inability of the host to elicit neutralizing antibodies to the newly emerging viral escape variants. Escape from autologous neutralizing activity was not associated with a reduction in the viral replication rate in vitro. Escape from autologous serum with cross-reactive neutralizing activity coincided with an increase in the length of the variable loops and in the number of potential N-linked glycosylation sites in the viral envelope. Positive selection pressure was observed in the variable regions in envelope, suggesting that, at least in these individuals, these regions are targeted by humoral immunity with cross-reactive potential. Our results may imply that the ability of HIV-1 to rapidly escape cross-reactive autologous neutralizing antibody responses without the loss of viral fitness is the underlying explanation for the absent effect of potent cross-reactive neutralizing humoral immunity on the clinical course of infection.
Previous clinical efficacy trials failed to support the continued development of recombinant gp120 (rgp120) as a candidate HIV vaccine. However, the recent RV144 HIV vaccine trial in Thailand showed that a prime/boost immunization strategy involving priming with canarypox vCP1521 followed by boosting with rgp120 could provide significant, although modest, protection from HIV infection. Based on these results, there is renewed interest in the development of rgp120 based antigens for follow up vaccine trials, where this immunization approach can be applied to other cohorts at high risk for HIV infection. Of particular interest are cohorts in Africa, India, and China that are infected with clade C viruses.
A panel of 10 clade C rgp120 envelope proteins was expressed in 293 cells, purified by immunoaffinity chromatography, and used to immunize guinea pigs. The resulting sera were collected and analyzed in checkerboard experiments for rgp120 binding, V3 peptide binding, and CD4 blocking activity. Virus neutralization studies were carried out with two different assays and two different panels of clade C viruses. A high degree of cross reactivity against clade C and clade B viruses and viral proteins was observed. Most, but not all of the immunogens tested elicited antibodies that neutralized tier 1 clade B viruses, and some sera neutralized multiple clade C viruses. Immunization with rgp120 from the CN97001 strain of HIV appeared to elicit higher cross neutralizing antibody titers than the other antigens tested.
While all of the clade C antigens tested were immunogenic, some were more effective than others in eliciting virus neutralizing antibodies. Neutralization titers did not correlate with rgp120 binding, V3 peptide binding, or CD4 blocking activity. CN97001 rgp120 elicited the highest level of neutralizing antibodies, and should be considered for further HIV vaccine development studies.
We screened 150 individuals from two recent seroconverter cohorts and found that six (4%) had CXCR4-using viruses. Clonal analysis of these six individuals, along with a seventh individual identified during clinical care as a recent seroconverter, revealed the presence of both X4- and dual-tropic variants in these recently infected adults. The ability of individual CXCR4-using variants to infect cells expressing CD4/CXCR4 or CD4/CCR5 varied dramatically. These data demonstrate that virus populations in some newly infected individuals can consist of either heterogeneous populations containing both CXCR4-using and CCR5-tropic viruses, or homogeneous populations containing only CXCR4-using viruses. The presence of CXCR4-using viruses at early stages of infection suggests that testing for viral tropism before using CCR5 antagonists may be important even in persons with known recent infection. The presence of CXCR4-using viruses in a subset of newly infected individuals could impact the efficacies of vaccine and microbicide strategies that target CCR5-tropic viruses.
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.
The identification of the determinants of sensitivity and resistance to broadly neutralizing antibodies is a high priority for human immunodeficiency virus (HIV) research. An analysis of the swarm of closely related envelope protein variants in an HIV-infected individual revealed a mutation that markedly affected sensitivity to neutralization by antibodies and antiviral entry inhibitors targeting both gp41 and gp120. This mutation mapped to the C34 helix of gp41 and disrupted an unexplored structural feature consisting of a ring of hydrogen bonds in the gp41 trimer. This mutation appeared to affect the assembly of the six-helix bundle required for virus fusion and to alter the conformational equilibria so as to favor the prehairpin intermediate conformation required for the binding of the membrane proximal external region-specific neutralizing antibodies 2F5 and 4E10 and the antiviral drug enfuvirtide (Fuzeon). The “swarm analysis” method we describe furthers our understanding of the relationships among the structure, function, and antigenicity of the HIV envelope protein and represents a new approach to the identification of vaccine antigens.
The development of a rapid and efficient system to identify human immunodeficiency virus type 1 (HIV-1)-infected individuals with broad and potent HIV-1-specific neutralizing antibody responses is an important step toward the discovery of critical neutralization targets for rational AIDS vaccine design. In this study, samples from HIV-1-infected volunteers from diverse epidemiological regions were screened for neutralization responses using pseudovirus panels composed of clades A, B, C, and D and circulating recombinant forms (CRFs). Initially, 463 serum and plasma samples from Australia, Rwanda, Uganda, the United Kingdom, and Zambia were screened to explore neutralization patterns and selection ranking algorithms. Samples were identified that neutralized representative isolates from at least four clade/CRF groups with titers above prespecified thresholds and ranked based on a weighted average of their log-transformed neutralization titers. Linear regression methods selected a five-pseudovirus subset, representing clades A, B, and C and one CRF01_AE, that could identify top-ranking samples with 50% inhibitory concentration (IC50) neutralization titers of ≥100 to multiple isolates within at least four clade groups. This reduced panel was then used to screen 1,234 new samples from the Ivory Coast, Kenya, South Africa, Thailand, and the United States, and 1% were identified as elite neutralizers. Elite activity is defined as the ability to neutralize, on average, more than one pseudovirus at an IC50 titer of 300 within a clade group and across at least four clade groups. These elite neutralizers provide promising starting material for the isolation of broadly neutralizing monoclonal antibodies to assist in HIV-1 vaccine design.
The V3 loop of HIV-1 gp120 is considered occluded on many primary viruses. However, virus sensitivity to neutralization by different V3 mAbs often varies, indicating that access to V3 is not restricted equally for all antibodies. Here, we have sought to gain a better understanding of these restrictions by determining the neutralizing activities of 7 V3 mAbs (19b, 39F, CO11, F2A3, F530, LA21, and LE311) against 15 subtype B primary isolates and relating these activities to the fine specificity of the mAbs. Not surprisingly, we found that most mAbs neutralized the same 2–3 viruses, with only mAb F530 able to neutralize 2 additional viruses not neutralized by the other mAbs. Epitope mapping revealed that positively-charged residues in or near the V3 stem are important for the binding of all the mAbs and that most mAbs seem to require the Pro residue that forms the GPGR β hairpin turn in the V3 tip for binding. Based on the mapping, we determined that V3 sequence variation accounted for neutralization resistance of approximately half the viruses tested. Comparison of these results to those of select V3 mAbs with overall better neutralizing activities in the light of structural information suggests that an antibody’s mode of interaction with V3, driven by contact residue requirements, precludes the antibody from accessing its epitope on different viruses. Based on the data we propose an angle of interaction with V3 that is less stringent on access for antibodies with cross-neutralizing activity compared to antibodies that neutralize relatively fewer viruses.
V3 antibodies; cross-neutralization; V3 accessibility; epitope mapping; HIV vaccine design
In efforts to develop AIDS vaccine components, we generated combinatorial libraries of recombinant human rhinoviruses that display the well-conserved ELDKWA epitope of the membrane-proximal external region of human immunodeficiency virus type 1 (HIV-1) gp41. The broadly neutralizing human monoclonal antibody 2F5 was used to select for viruses whose ELDKWA conformations resemble those of HIV. Immunization of guinea pigs with different chimeras, some boosted with ELDKWA-based peptides, elicited antibodies capable of neutralizing HIV-1 pseudoviruses of diverse subtypes and coreceptor usages. These recombinant immunogens are the first reported that elicit broad, albeit modest, neutralization of HIV-1 using an ELDKWA-based epitope and are among the few reported that elicit broad neutralization directed against any recombinant HIV epitope, providing a critical advance in developing effective AIDS vaccine components.
Studies were performed to induce cross-clade neutralizing antibodies (Abs) by testing various combinations of prime and boost constructs that focus the immune response on structurally conserved epitopes in the V3 loop of HIV-1 gp120. Rabbits were immunized with gp120 DNA containing a V3 loop characterized by the GPGR motif at its tip, and/or with gp120 DNA with a V3 loop carrying the GPGQ motif. Priming was followed by boosts with a V3-fusion proteins (V3-FPs) carrying the V3 sequence from a subtype B virus (GPGR motif), and/or with V3 sequences from subtypes A and C (GPGQ motif). The broadest and most consistent neutralizing responses were generated when using a clade C gp120 DNA prime and with the V3B-FP boost. Immune sera displayed neutralizing activity in three assays against pseudoviruses and primary isolates from subtypes A, AG, B, C, and D. Polyclonal Abs in the immune rabbit sera neutralized viruses that were not neutralized by pools of human anti-V3 monoclonal Abs. Greater than 80% of the neutralizing Abs were specific for V3, showing that the immune response could be focused on a neutralizing epitope and that vaccine-induced anti-V3 Abs have cross-clade neutralizing activity.
HIV-1; Vaccine; Neutralizing antibodies; V3 loop; gp120; Pseudovirus; Immunization