HIV-1 gp41

What is the basic structure and function of HIV-1 gp41 in viral entry?

HIV-1 gp41 is one of two non-covalently associated subunits of the HIV envelope glycoprotein (Env), with gp120 being the other. While gp120 is responsible for binding to cell surface-associated receptor (CD4) and coreceptor (CCR5 or CXCR4), gp41 mediates the fusion of the viral membrane with the plasma cell membrane . This fusion process delivers the viral genome into the host cell, initiating the infection cycle .

Which regions of gp41 demonstrate evolutionary conservation despite HIV's high mutation rate?

Despite the significant sequence variation observed in HIV-1 Env, certain regions of gp41 show remarkable conservation across viral subtypes. The N-terminal heptad repeat (N-HR) and C-terminal heptad repeat (C-HR) regions that form the six-helix bundle critical for membrane fusion maintain substantial conservation . Notably, a hydrophobic cavity in the N-HR trimer that interacts with C-HR helices contains residues that are identical between HIV-1 and SIV strains, indicating strong evolutionary pressure to maintain this structure .

Analysis of sequence alignments across HIV-1 subtypes reveals that while variable regions may show 60-80% amino acid variability, other regions contain nearly invariant residues, suggesting functional constraints that prevent mutation without significant loss of viral fitness .

How do antibody responses to gp41 develop during acute HIV-1 infection?

During acute HIV-1 infection, initial antibody responses typically arise approximately 13 days after the onset of viremia and are primarily targeted to gp41 rather than gp120 . These early gp41 antibodies are generally non-neutralizing and ineffective at controlling viremia . The first antibodies capable of selecting viral escape mutants are gp120 autologous neutralizing antibodies, which appear only months after transmission .

Plasma cell analysis during acute HIV-1 infection reveals that approximately 6.5 ± 2.8% of total B cells are plasma cells . Among antibodies isolated from these plasma cells, 6.2% were reactive with gp41, while only 0.2% were reactive with gp120 . The somatic mutation frequencies in these gp41-reactive antibodies are relatively high (mean VH mutation frequency of 5.0 ± 0.4%), suggesting they may not represent a true primary immune response .

What is the origin of gp41-reactive antibodies in acute HIV-1 infection?

Two possible origins have been proposed for the gp41-reactive, highly mutated B cells recovered from acutely HIV-infected individuals:

• HIV-1 gp41 Env may trigger naive B cells to proliferate and rapidly accumulate mutations. This scenario is considered less plausible given the short timeframe between HIV-1 transmission and antibody detection (typically 17-30 days), especially considering the CD4+ T cell depletion and disruption of mucosal germinal centers that occur in acute HIV infection .

• More likely, HIV-1 gp41 activates pre-existing mutated B cells that cross-react with gp41. Studies of reverted unmutated ancestors (RUAs) of gp41-reactive antibodies show that many do not bind to HIV-1 Env but instead are polyreactive and bind to host or bacterial antigens . This suggests that many gp41 antibodies originate from memory B cells previously activated by non-HIV antigens.

The second hypothesis is supported by evidence from one large clonal lineage (lineage 558) in which reactivity to HIV-1 Env was acquired only after somatic mutations .

What are the characteristics of mucosal gp41-specific antibodies in HIV-1 infection?

The appearance of these antibodies is preceded by elevation of B-cell-activating factor belonging to the TNF family (BAFF), which likely contributes to an initial T-independent antibody response . The transient nature of these mucosal antibodies suggests they may not provide durable protection against HIV, which has important implications for vaccine design strategies targeting mucosal immunity.

How can researchers isolate and characterize gp41-specific antibodies from infected individuals?

Researchers employ several complementary techniques to isolate and characterize gp41-specific antibodies:

• Flow cytometry sorting of CD19+, CD27hi, CD38hi, and CD20lo or CD20- plasmablasts/plasma cells from blood or bone marrow of acutely infected individuals .

• RT-PCR amplification of rearranged variable regions of Ig heavy and light chain genes (VH and VL) from sorted cells .

• Production of recombinant monoclonal antibodies by cloning these sequences into expression vectors.

• Screening of antibodies against various HIV-1 Env proteins, including autologous HIV-1 gp140 proteins derived from the transmitted/founder virus, group M consensus Env gp140, and recombinant gp41 .

• Characterization of binding properties using multiple assays including ELISA, Luminex, and surface plasmon resonance (SPR) .

This methodological approach has enabled the identification of multiple clonal lineages of gp41-reactive antibodies within acutely infected individuals. For example, one study identified 35 clonal lineages among 977 plasma cell-derived antibodies, with 12 lineages containing members reactive with HIV-1 gp41 .

What techniques are used to study antibody affinity maturation to gp41?

To study antibody affinity maturation to gp41, researchers use a combination of experimental and computational approaches:

• Generation of reverted unmutated ancestors (RUAs) and inferred intermediate antibodies by computational analysis of somatic mutations .

• Expression of these constructs as recombinant antibodies for functional analysis.

• Evaluation of binding affinities using surface plasmon resonance (SPR) and Luminex assays to track changes in affinity as mutations accumulate .

• Deep sequencing (454 sequencing) of V(D)J gene segments from genomic DNAs to identify and track the evolution of antibody clonal lineages .

These approaches allow researchers to reconstruct the developmental pathway of gp41-specific antibodies. For example, in clone lineage 558, the affinity of antibody binding to rgp41 increased with accumulation of mutations, with Kd values improving from 63.3 nM at an early intermediate to 0.6 nM at a more mature observed antibody .

How can researchers express and purify recombinant gp41 for structural and immunological studies?

Expression and purification of recombinant gp41 present specific challenges due to the hydrophobic nature of certain domains. Common approaches include:

• Expression of gp41 fragments (particularly the ectodomain) in bacterial systems, often as fusion proteins with solubility-enhancing tags.

• Mammalian cell expression systems for producing glycosylated gp41 that maintains native post-translational modifications, which can be important for immunological studies.

• Stabilization strategies incorporating trimerization domains to maintain the native trimeric structure of gp41.

• Multi-step purification protocols typically involving affinity chromatography followed by size exclusion and/or ion exchange chromatography.

• Quality control using biophysical techniques such as circular dichroism, thermal denaturation, and analytical ultracentrifugation to confirm proper folding and oligomeric state.

For structural studies focusing on specific domains like the six-helix bundle, researchers have successfully used peptide synthesis or bacterial expression of individual heptad repeat regions .

How do gp41-specific antibody responses differ between acute infection and vaccination?

Several key differences have been observed between antibody responses elicited by natural infection versus vaccination:

• Mutation frequency: Antibodies isolated from acutely infected individuals show higher mutation frequencies (mean VH mutation frequency of 5.0 ± 0.4%) compared to those elicited after primary HIV-1 Env vaccination (1.5 ± 0.5% at day 42 post-vaccination) .

• Unmutated antibodies: A lower percentage of unmutated HIV-1-reactive antibodies is found in acute infection (10.4%, all gp41 reactive) compared to primary HIV-1 Env vaccination (28.6%) .

• Affinity maturation: After vaccination, antibody affinity increases more gradually over time, reaching similar mutation levels (4.8 ± 0.5%) only after multiple immunizations (by day 182 after 4 Env immunizations) .

These differences suggest that natural infection and vaccination may stimulate distinct B cell populations and developmental pathways, which has important implications for HIV vaccine design strategies.

What are the potential mechanisms for the short half-life of mucosal gp41-specific IgA antibodies?

The remarkably short half-life (approximately 2.7 days) observed for mucosal gp41-specific IgA antibodies raises important questions about the mechanisms controlling antibody persistence at mucosal surfaces . Several hypotheses might explain this phenomenon:

• Regulation of plasma cell survival in mucosal tissues during acute infection.

• Changes in expression of homing receptors on antibody-secreting cells.

• Alterations in local cytokine environment affecting antibody production and secretion.

• Increased degradation or clearance of antibodies at mucosal surfaces during inflammation.

Understanding these mechanisms is critical for developing vaccination strategies that can induce durable mucosal antibody responses, which may be necessary for preventing HIV-1 transmission at mucosal surfaces .

How does clonal lineage development of gp41-specific antibodies evolve from acute to chronic infection?

The evolution of gp41-specific antibody lineages over time provides insights into how the immune response to HIV develops. Studies using deep sequencing of B cell receptor repertoires have identified gp41-reactive clonal lineages present during acute infection that can be tracked longitudinally .

Analysis of one particularly large clonal family (lineage 558 from subject 684-6) containing 51 members demonstrated how somatic hypermutation accumulated over time, with corresponding increases in binding affinity to gp41 . The identification of intermediate forms through deep sequencing allows researchers to reconstruct the evolutionary pathway of these antibodies and understand how HIV reactivity develops during infection.

This approach has revealed that some gp41 lineages initially lack reactivity to HIV but acquire it through somatic mutation, supporting the hypothesis that these antibodies originate from B cells initially triggered by non-HIV antigens .

What conserved structural features of gp41 could serve as targets for broadly neutralizing antibodies?

Despite HIV's remarkable variability, several conserved structural features of gp41 represent potential targets for broadly neutralizing antibodies:

• The hydrophobic cavity in the N-terminal heptad repeat (N-HR) trimer that interacts with C-terminal heptad repeat (C-HR) helices. The residues forming this cavity are identical between HIV-1 and SIV strains .

• Regions critical for the formation and stability of the six-helix bundle that mediates membrane fusion. Mutations in these regions affect HIV infectivity and fusion capability .

• The membrane-proximal external region (MPER), which contains epitopes recognized by some broadly neutralizing antibodies.

These conserved structures may have limited capacity for escape mutations without compromising viral fitness, making them attractive targets for therapeutic antibodies or small molecule inhibitors .

How can understanding early gp41 antibody responses inform HIV vaccine design?

The characteristics of early gp41 antibody responses have several implications for HIV vaccine design:

• The finding that many early gp41 antibodies arise from pre-existing memory B cells that cross-react with non-HIV antigens suggests that these responses may be readily elicited but potentially less protective .

• The short half-life of mucosal gp41-specific IgA antibodies indicates that strategies to extend antibody durability at mucosal surfaces may be critical for protective vaccines .

• The observation that natural infection and vaccination elicit antibodies with different mutation frequencies and developmental pathways suggests that vaccine strategies may need to be optimized to target specific B cell populations .

• Understanding the conserved structural features of gp41 that are critical for viral function can guide the design of immunogens that focus the immune response on these vulnerable sites .