Intrasubtype B HIV-1 Superinfection Correlates with Delayed Neutralizing Antibody Response.

Understanding whether the neutralizing antibody (NAb) response impacts HIV-1 superinfection and how superinfection subsequently modulates the NAb response can help clarify correlates of protection from HIV exposures and better delineate pathways of NAb development. We examined associations between the development of NAb and the occurrence of superinfection in a well-characterized, antiretroviral therapy (ART)-naive, primary infection cohort of men who have sex with men. Deep sequencing was applied to blood plasma samples from the cohort to detect cases of superinfection. We compared the NAb activity against autologous and heterologous viruses between 10 participants with intrasubtype B superinfection and 19 monoinfected controls, matched to duration of infection and risk behavior. Three to 6 months after primary infection, individuals who would later become superinfected had significantly weaker NAb activity against tier 1 subtype B viruses (P = 0.003 for SF-162 and P = 0.017 for NL4-3) and marginally against autologous virus (P = 0.054). Lower presuperinfection NAb responses correlated with weaker gp120 binding and lower plasma total IgG titers. Soon after superinfection, the NAb response remained lower, but between 2 and 3 years after primary infection, NAb levels strengthened and reached those of controls. Superinfecting viruses were typically not susceptible to neutralization by presuperinfection plasma. These observations suggest that recently infected individuals with a delayed NAb response against primary infecting and tier 1 subtype B viruses are more susceptible to superinfection.IMPORTANCE Our findings suggest that within the first year after HIV infection, a relatively weak neutralizing antibody response against primary and subtype-specific neutralization-sensitive viruses increases susceptibility to superinfection in the face of repeated exposures. As natural infection progresses, the immune response strengthens significantly in some superinfected individuals. These findings will inform HIV vaccine design by providing testable correlates of protection from initial HIV infection.

Broadly Neutralizing Antibody Responses in a Large Longitudinal Sub-Saharan HIV Primary Infection Cohort.

Broadly neutralizing antibodies (bnAbs) are thought to be a critical component of a protective HIV vaccine. However, designing vaccines immunogens able to elicit bnAbs has proven unsuccessful to date. Understanding the correlates and immunological mechanisms leading to the development of bnAb responses during natural HIV infection is thus critical to the design of a protective vaccine. The IAVI Protocol C program investigates a large longitudinal cohort of primary HIV-1 infection in Eastern and South Africa. Development of neutralization was evaluated in 439 donors using a 6 cross-clade pseudo-virus panel predictive of neutralization breadth on larger panels. About 15% of individuals developed bnAb responses, essentially between year 2 and year 4 of infection. Statistical analyses revealed no influence of gender, age or geographical origin on the development of neutralization breadth. However, cross-clade neutralization strongly correlated with high viral load as well as with low CD4 T cell counts, subtype-C infection and HLA-A*03(-) genotype. A correlation with high overall plasma IgG levels and anti-Env IgG binding titers was also found. The latter appeared not associated with higher affinity, suggesting a greater diversity of the anti-Env responses in broad neutralizers. Broadly neutralizing activity targeting glycan-dependent epitopes, largely the N332-glycan epitope region, was detected in nearly half of the broad neutralizers while CD4bs and gp41-MPER bnAb responses were only detected in very few individuals. Together the findings suggest that both viral and host factors are critical for the development of bnAbs and that the HIV Env N332-glycan supersite may be a favorable target for vaccine design.

Broad neutralization coverage of HIV by multiple highly potent antibodies.

Broadly neutralizing antibodies against highly variable viral pathogens are much sought after to treat or protect against global circulating viruses. Here we probed the neutralizing antibody repertoires of four human immunodeficiency virus (HIV)-infected donors with remarkably broad and potent neutralizing responses and rescued 17 new monoclonal antibodies that neutralize broadly across clades. Many of the new monoclonal antibodies are almost tenfold more potent than the recently described PG9, PG16 and VRC01 broadly neutralizing monoclonal antibodies and 100-fold more potent than the original prototype HIV broadly neutralizing monoclonal antibodies. The monoclonal antibodies 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 broadly neutralizing monoclonal antibodies now available reveals that certain combinations of antibodies should offer markedly more favourable 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 broadly neutralizing monoclonal antibodies 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.

Predicting HIV-1 broadly neutralizing antibody epitope networks using neutralization titers and a novel computational method.

BACKGROUND: Recent efforts in HIV-1 vaccine design have focused on immunogens that evoke potent neutralizing antibody responses to a broad spectrum of viruses circulating worldwide. However, the development of effective vaccines will depend on the identification and characterization of the neutralizing antibodies and their epitopes. We developed bioinformatics methods to predict epitope networks and antigenic determinants using structural information, as well as corresponding genotypes and phenotypes generated by a highly sensitive and reproducible neutralization assay.282 clonal envelope sequences from a multiclade panel of HIV-1 viruses were tested in viral neutralization assays with an array of broadly neutralizing monoclonal antibodies (mAbs: b12, PG9,16, PGT121 - 128, PGT130 - 131, PGT135 - 137, PGT141 - 145, and PGV04). We correlated IC50 titers with the envelope sequences, and used this information to predict antibody epitope networks. Structural patches were defined as amino acid groups based on solvent-accessibility, radius, atomic depth, and interaction networks within 3D envelope models. We applied a boosted algorithm consisting of multiple machine-learning and statistical models to evaluate these patches as possible antibody epitope regions, evidenced by strong correlations with the neutralization response for each antibody. RESULTS: We identified patch clusters with significant correlation to IC50 titers as sites that impact neutralization sensitivity and therefore are potentially part of the antibody binding sites. Predicted epitope networks were mostly located within the variable loops of the envelope glycoprotein (gp120), particularly in V1/V2. Site-directed mutagenesis experiments involving residues identified as epitope networks across multiple mAbs confirmed association of these residues with loss or gain of neutralization sensitivity. CONCLUSIONS: Computational methods were implemented to rapidly survey protein structures and predict epitope networks associated with response to individual monoclonal antibodies, which resulted in the identification and deeper understanding of immunological hotspots targeted by broadly neutralizing HIV-1 antibodies.

Dynamics of viral evolution and neutralizing antibody response after HIV-1 superinfection.

Investigating the incidence and prevalence of HIV-1 superinfection is challenging due to the complex dynamics of two infecting strains. The superinfecting strain can replace the initial strain, be transiently expressed, or persist along with the initial strain in distinct or in recombined forms. Various selective pressures influence these alternative scenarios in different HIV-1 coding regions. We hypothesized that the potency of the neutralizing antibody (NAb) response to autologous viruses would modulate viral dynamics in env following superinfection in a limited set of superinfection cases. HIV-1 env pyrosequencing data were generated from blood plasma collected from 7 individuals with evidence of superinfection. Viral variants within each patient were screened for recombination, and viral dynamics were evaluated using nucleotide diversity. NAb responses to autologous viruses were evaluated before and after superinfection. In 4 individuals, the superinfecting strain replaced the original strain. In 2 individuals, both initial and superinfecting strains continued to cocirculate. In the final individual, the surviving lineage was the product of interstrain recombination. NAb responses to autologous viruses that were detected within the first 2 years of HIV-1 infection were weak or absent for 6 of the 7 recently infected individuals at the time of and shortly following superinfection. These 6 individuals had detectable on-going viral replication of distinct superinfecting virus in the env coding region. In the remaining case, there was an early and strong autologous NAb response, which was associated with extensive recombination in env between initial and superinfecting strains. This extensive recombination made superinfection more difficult to identify and may explain why the detection of superinfection has typically been associated with low autologous NAb titers.

Identification of an HIV-1 clade A envelope that exhibits broad antigenicity and neutralization sensitivity and elicits antibodies targeting three distinct epitopes.

Broadly neutralizing antibodies (bNAbs) PG9 and PG16 were isolated from an International AIDS Vaccine Initiative (IAVI) Protocol G subject infected with human immunodeficiency virus type 1 (HIV-1) clade A. Both antibodies are highly potent and neutralize greater than 70% of viruses tested. We sought to begin immunogen design based on viral sequences from this patient; however, pseudoviruses prepared with 19 envelope sequences from this subject were resistant to neutralization by PG9 and PG16. Therefore, we used a bioinformatics approach to identify closely related viruses that were potentially sensitive to PG9 and PG16. A most-recent common ancestor (MRCA) sequence for the viral envelope (Env) was determined and aligned with 99 subtype A gp160 sequences from the Los Alamos HIV database. Virus BG505.W6M.ENV.C2 (BG505) was found to have the highest degree of homology (73%) to the MRCA sequence. Pseudoviruses prepared with this Env were sensitive to neutralization with a broad panel of bNAbs, including PG9 and PG16. When expressed by 293T cells as soluble gp120, the BG505 monomer bound well to both PG9 and PG16. We further showed that a point mutation (L111A) enabled more efficient production of a stable gp120 monomer that preserves the major neutralization epitopes. Finally, we showed that an adjuvanted formulation of this gp120 protein elicited neutralizing antibodies in rabbits (following a gp120 DNA vaccine prime) and that the antisera competed with bNAbs from 3 classes of nonoverlapping epitopes. Thus, the BG505 Env protein warrants further investigation as an HIV vaccine candidate, as a stand-alone protein, or as a component of a vaccine vector.

Longitudinal analysis of early HIV-1-specific neutralizing activity in an elite neutralizer and in five patients who developed cross-reactive neutralizing activity.

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.

Sequences in glycoprotein gp41, the CD4 binding site, and the V2 domain regulate sensitivity and resistance of HIV-1 to broadly neutralizing antibodies.

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.

Mutation at a single position in the V2 domain of the HIV-1 envelope protein confers neutralization sensitivity to a highly neutralization-resistant virus.

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 α4ß7 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 α4ß7.

Cross-reactive neutralizing humoral immunity does not protect from HIV type 1 disease progression.

Broadly reactive neutralizing antibodies are the focus of human immunodeficiency virus (HIV) type 1 vaccine design. However, only little is known about their role in acquired immunodeficiency syndrome (AIDS) pathogenesis and the factors associated with their development. Here we used a multisubtype panel of 23 HIV-1 variants to determine the prevalence of cross-reactive neutralizing activity in serum samples obtained approximately 35 months after seroconversion from 82 HIV-1 subtype B-infected participants from the Amsterdam Cohort Studies on HIV Infection and AIDS. Of these patients, 33%, 48%, and 20%, respectively, had strong, moderate, or absent cross-reactive neutralizing activity in serum. Viral RNA load at set point and AIDS-free survival were similar for the 3 patient groups. However, higher cross-reactive neutralizing activity was significantly associated with lower CD4(+) T cell counts before and soon after infection. Our findings underscore the importance of vaccine-elicited immunity in protecting from infection. The association between CD4(+) T cell counts and neutralizing humoral immunity may provide new clues as to how to achieve this goal.

Broad neutralization of human immunodeficiency virus type 1 (HIV-1) elicited from human rhinoviruses that display the HIV-1 gp41 ELDKWA epitope.

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.

Changing the thickness of two layers: i-ZnO nanorods, p-Cu2O and its influence on the carriers transport mechanism of the p-Cu2O/i-ZnO nanorods/n-IGZO heterojunction.

In this study, two layers: i-ZnO nanorods and p-Cu2O were fabricated by electrochemical deposition. The fabricating process was the initial formation of ZnO nanorods layer on the n-IGZO thin film which was prepared by sputtering method, then a p-Cu2O layer was deposited on top of rods to form the p-Cu2O/i-ZnO nanorods/n-ZnO heterojunction. The XRD, SEM, UV-VIS, I-V characteristics methods were used to define structure, optical and electrical properties of these heterojunction layers. The fabricating conditions and thickness of the Cu2O layers significantly affected to the formation, microstructure, electrical and optical properties of the junction. The length of i-ZnO nanorods layer in the structure of the heterojunction has strongly affected to the carriers transport mechanism and performance of this heterojunction.

Rapid Sequencing of Complete env Genes from Primary HIV-1 Samples.

The ability to study rapidly evolving viral populations has been constrained by the read length of next-generation sequencing approaches and the sampling depth of single-genome amplification methods. Here, we develop and characterize a method using Pacific Biosciences' Single Molecule, Real-Time (SMRT®) sequencing technology to sequence multiple, intact full-length human immunodeficiency virus-1 env genes amplified from viral RNA populations circulating in blood, and provide computational tools for analyzing and visualizing these data.

HIV-1 neutralizing antibody response and viral genetic diversity characterized with next generation sequencing.

To better understand the dynamics of HIV-specific neutralizing antibody (NAb), we examined associations between viral genetic diversity and the NAb response against a multi-subtype panel of heterologous viruses in a well-characterized, therapy-naïve primary infection cohort. Using next generation sequencing (NGS), we computed sequence-based measures of diversity within HIV-1 env, gag and pol, and compared them to NAb breadth and potency as calculated by a neutralization score. Contemporaneous env diversity and the neutralization score were positively correlated (p=0.0033), as were the neutralization score and estimated duration of infection (EDI) (p=0.0038), and env diversity and EDI (p=0.0005). Neither early env diversity nor baseline viral load correlated with future NAb breadth and potency (p>0.05). Taken together, it is unlikely that neutralizing capability in our cohort was conditioned on viral diversity, but rather that env evolution was driven by the level of NAb selective pressure.

Five Antiretroviral Drug Class-Resistant HIV-1 in a Treatment-Naïve Patient Successfully Suppressed with Optimized Antiretroviral Drug Selection.

Transmitted HIV-1 exhibiting reduced susceptibility to protease and reverse transcriptase inhibitors is well documented but limited for integrase inhibitors and enfuvirtide. We describe here a case of transmitted 5 drug class-resistance in an antiretroviral (ARV)-naïve patient who was successfully treated based on the optimized selection of an active ARV drug regimen. The value of baseline resistance testing to determine an optimal ARV treatment regimen is highlighted in this case report.

Glycans flanking the hypervariable connecting peptide between the A and B strands of the V1/V2 domain of HIV-1 gp120 confer resistance to antibodies that neutralize CRF01_AE viruses.

Understanding the molecular determinants of sensitivity and resistance to neutralizing antibodies is critical for the development of vaccines designed to prevent HIV infection. In this study, we used a genetic approach to characterize naturally occurring polymorphisms in the HIV envelope protein that conferred neutralization sensitivity or resistance. Libraries of closely related envelope genes, derived from virus quasi-species, were constructed from individuals infected with CRF01_AE viruses. The libraries were screened with plasma containing broadly neutralizing antibodies, and neutralization sensitive and resistant variants were selected for sequence analysis. In vitro mutagenesis allowed us to identify single amino acid changes in three individuals that conferred resistance to neutralization by these antibodies. All three mutations created N-linked glycosylation sites (two at N136 and one at N149) proximal to the hypervariable connecting peptide between the C-terminus of the A strand and the N-terminus of the B strand in the four-stranded V1/V2 domain ß-sheet structure. Although N136 has previously been implicated in the binding of broadly neutralizing monoclonal antibodies, this glycosylation site appears to inhibit the binding of neutralizing antibodies in plasma from HIV-1 infected subjects. Previous studies have reported that the length of the V1/V2 domain in transmitted founder viruses is shorter and possesses fewer glycosylation sites compared to viruses isolated from chronic infections. Our results suggest that vaccine immunogens based on recombinant envelope proteins from clade CRF01_AE viruses might be improved by inclusion of envelope proteins that lack these glycosylation sites. This strategy might improve the efficacy of the vaccines used in the partially successful RV144 HIV vaccine trial, where the two CRF01_AE immunogens (derived from the A244 and TH023 isolates) both possessed glycosylation sites at N136 and N149.

Highly pathogenic adapted HIV-1 strains limit host immunity and dictate rapid disease progression.

OBJECTIVE: The study of HIV-1 rapid progressors has been limited to specific case reports. Nevertheless, identification and characterization of the viral and host factors involved in rapid progression are crucial when attempting to uncover the correlates of rapid disease outcome. DESIGN: We carried out comparative functional analyses in rapid progressors (n = 46) and standard progressors (n = 46) early after HIV-1 seroconversion (≤1 year). The viral traits tested were viral replicative capacity, co-receptor usage, and genomic variation. Host CD8(+) T-cell responses, humoral activity, and HLA immunogenetic markers were also determined. RESULTS: Our data demonstrate an unusual convergence of highly pathogenic HIV-1 strains in rapid progressors. Compared with standard progressors, rapid progressor viral strains show higher in-vitro replicative capacity (81.5 vs. 67.9%; P = 0.025) and greater X4/DM co-receptor usage (26.3 vs. 2.8%; P = 0.006) in early infection. Limited or absent functional HIV-1 CD8(+) T-cell responses and neutralizing activity were measured in rapid progressors. Moreover, the increase in common HLA allele-restricted CD8(+) T-cell escape mutations in rapid progressors acts as a signature of uncontrolled HIV-1 replication and early impairment of adaptive cellular responses. CONCLUSION: Our data support a dominant role for viral factors in rapid progressors. Robust HIV-1 replication and intrinsic viral properties limit host adaptive immune responses, thus driving rapid disease progression.

Prime-boost immunization of rabbits with HIV-1 gp120 elicits potent neutralization activity against a primary viral isolate.

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 ∼10(3) to 10(4) 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.

Combined antiretroviral therapy and immune pressure lead to in vivo HIV-1 recombination with ancestral viral genomes.

BACKGROUND: Studies on drug interruption have provided new insights on the adaptive evolution of rebounding HIV-1 during antiretroviral pressure. We investigated the origin of new viral variants after discontinuation of protease (PR) inhibitors as a treatment remained exclusively based on reverse transcriptase inhibitors, and whether drug susceptibility, viral fitness, and neutralizing antibodies could be major driving forces for the evolution of virus populations. METHODS: The study comprised 3 treatment-experienced subjects. Phylogenetic analysis of the PR, reverse transcriptase, and the viral envelope were carried out to ascertain the origin of the new viral variants with samples obtained over a 10-year period before and after a PR inhibitor withdrawal. In addition, drug susceptibility, replication capacity, and neutralization assays were performed. RESULTS: New viral variants from all 3 subjects were derived through recombination with ancestral quasispecies. Computerized recombination models confirmed these results. Recombination was demonstrated by increased replication capacity, decreased drug susceptibility, and neutralization of ancestral virus envelope by contemporaneous plasma samples. CONCLUSIONS: These findings demonstrate the relevance of HIV-1 reservoirs in adaptive evolution throughout recombination in response to selective pressure, such as antiretroviral therapy and immune responses. This result might assist in the design of new treatment strategies for patients experiencing treatment failure.

Effect of trimerization motifs on quaternary structure, antigenicity, and immunogenicity of a noncleavable HIV-1 gp140 envelope glycoprotein.

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 Escherichia coli aspartate transcarbamoylase catalytic subunit. Fusion of each motif to the C-terminus of a noncleavable 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.