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Association of VH4-59 Antibody Variable Gene Usage with Recognition of an Immunodominant Epitope on the HIV-1 Gag Protein.

Chukwuma VU, Hicar MD, Chen X, Nicholas KJ, Joyner A, Kalams SA, Landucci G, Forthal DN, Spearman PW, Crowe JE - PLoS ONE (2015)

Bottom Line: We found no evidence that mAb 3E4 exhibited any function in laboratory studies aimed at elucidating the immunologic activity, including assays for neutralization, Ab-dependent cell-mediated virus inhibition, or enhanced T cell reactivity caused by Gag-3E4 complexes.The findings suggest this immunodominant epitope in Gag protein, which is associated with VH4-59 germline gene usage, may induce a high level of B cells that encode binding but non-functional antibodies that occupy significant repertoire space following HIV infection.The studies define an additional specific molecular mechanism in the immune distraction activity of the HIV virion.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Microbiology and Immunology, Vanderbilt University, Nashville, Tennessee, United States of America.

ABSTRACT
The human antibody response against HIV-1 infection recognizes diverse antigenic subunits of the virion, and includes a high level of antibodies to the Gag protein. We report here the isolation and characterization of a subset of Gag-specific human monoclonal antibodies (mAbs) that were prevalent in the antibody repertoire of an HIV-infected individual. Several lineages of Gag-specifc mAbs were encoded by a single antibody heavy chain variable region, VH4-59, and a representative antibody from this group designated mAb 3E4 recognized a linear epitope on the globular head of the p17 subunit of Gag. We found no evidence that mAb 3E4 exhibited any function in laboratory studies aimed at elucidating the immunologic activity, including assays for neutralization, Ab-dependent cell-mediated virus inhibition, or enhanced T cell reactivity caused by Gag-3E4 complexes. The findings suggest this immunodominant epitope in Gag protein, which is associated with VH4-59 germline gene usage, may induce a high level of B cells that encode binding but non-functional antibodies that occupy significant repertoire space following HIV infection. The studies define an additional specific molecular mechanism in the immune distraction activity of the HIV virion.

No MeSH data available.


Related in: MedlinePlus

Identifying HIV proteins recognized by mAb 3E4.(A) SDS-PAGE separation on a 4–12% gradient gel of Env VLP, Gag-only VLP, 293F, 293T, Jurkat or TZM-bl cell lysates, followed by Coomassie staining. (B) Western blot analysis of the cell lysates using mAb 3E4 as a probe. The molecular weight markers are indicated. (C) Western blot analysis of NL4-3 pelleted particles using mAbs CA183 (left panel) or 3E4 (right panel) as probes. Bands representing the molecular weights of p55 Gag, p24 CA and p17 MA are indicated.
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pone.0133509.g003: Identifying HIV proteins recognized by mAb 3E4.(A) SDS-PAGE separation on a 4–12% gradient gel of Env VLP, Gag-only VLP, 293F, 293T, Jurkat or TZM-bl cell lysates, followed by Coomassie staining. (B) Western blot analysis of the cell lysates using mAb 3E4 as a probe. The molecular weight markers are indicated. (C) Western blot analysis of NL4-3 pelleted particles using mAbs CA183 (left panel) or 3E4 (right panel) as probes. Bands representing the molecular weights of p55 Gag, p24 CA and p17 MA are indicated.

Mentions: The occurrence of a single heavy chain variable gene segment encoding antibodies with the Gag-only VLP binding specificity suggested that the binding might not just be nonspecific interaction of diverse B cells to varying components of the VLPs, but rather a specific molecular interaction mediated by antibodies encoded by this gene segment. We sought to determine whether Gag-only VLP binding was due to specific interactions with host proteins from the cell membrane or to HIV internal proteins. We tested binding of mAb 3E4 as a representative mAb of this class to lysates made from several human cell lines or from Gag-only HIV VLPs. Lysates were electrophoresed on two 4–12% NuPAGE Bis-Tris gels, one of which was stained by Coomassie blue staining to verify the presence of proteins in the cell lysates (Fig 3A). In order to determine the size of any proteins in the cell lysates interacting specifically with mAb 3E4, we performed western blot analysis on the second gel after electrophoresis, using mAb 3E4 as a probe. We observed the presence of a band with a 55 kDa apparent molecular weight in the lysates from the VLPs, which was absent in the lysates from the human cell lines (Fig 3B). This finding indicated that mAb 3E4 was binding to a viral component of the VLPs, recognizing a band at an apparent molecular weight of 55 kDa that could be Gag because of the expected migration of p55 Gag. To test our hypothesis, we performed western blot analysis using cell lysates from NL4-3 virus produced in 293T or HeLa cell lines, using mAb 3E4 as a probe. As a positive control, we included capsid-specific mAb CA183 that binds to a region on the top of Gag (Fig 3C, left panel). The results showed that mAb 3E4 binds to p55 Gag and p17 Matrix proteins (Fig 3C, right panel). These findings revealed that the VH4-59 gene-encoded mAbs bind to the p17 Matrix protein.


Association of VH4-59 Antibody Variable Gene Usage with Recognition of an Immunodominant Epitope on the HIV-1 Gag Protein.

Chukwuma VU, Hicar MD, Chen X, Nicholas KJ, Joyner A, Kalams SA, Landucci G, Forthal DN, Spearman PW, Crowe JE - PLoS ONE (2015)

Identifying HIV proteins recognized by mAb 3E4.(A) SDS-PAGE separation on a 4–12% gradient gel of Env VLP, Gag-only VLP, 293F, 293T, Jurkat or TZM-bl cell lysates, followed by Coomassie staining. (B) Western blot analysis of the cell lysates using mAb 3E4 as a probe. The molecular weight markers are indicated. (C) Western blot analysis of NL4-3 pelleted particles using mAbs CA183 (left panel) or 3E4 (right panel) as probes. Bands representing the molecular weights of p55 Gag, p24 CA and p17 MA are indicated.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520566&req=5

pone.0133509.g003: Identifying HIV proteins recognized by mAb 3E4.(A) SDS-PAGE separation on a 4–12% gradient gel of Env VLP, Gag-only VLP, 293F, 293T, Jurkat or TZM-bl cell lysates, followed by Coomassie staining. (B) Western blot analysis of the cell lysates using mAb 3E4 as a probe. The molecular weight markers are indicated. (C) Western blot analysis of NL4-3 pelleted particles using mAbs CA183 (left panel) or 3E4 (right panel) as probes. Bands representing the molecular weights of p55 Gag, p24 CA and p17 MA are indicated.
Mentions: The occurrence of a single heavy chain variable gene segment encoding antibodies with the Gag-only VLP binding specificity suggested that the binding might not just be nonspecific interaction of diverse B cells to varying components of the VLPs, but rather a specific molecular interaction mediated by antibodies encoded by this gene segment. We sought to determine whether Gag-only VLP binding was due to specific interactions with host proteins from the cell membrane or to HIV internal proteins. We tested binding of mAb 3E4 as a representative mAb of this class to lysates made from several human cell lines or from Gag-only HIV VLPs. Lysates were electrophoresed on two 4–12% NuPAGE Bis-Tris gels, one of which was stained by Coomassie blue staining to verify the presence of proteins in the cell lysates (Fig 3A). In order to determine the size of any proteins in the cell lysates interacting specifically with mAb 3E4, we performed western blot analysis on the second gel after electrophoresis, using mAb 3E4 as a probe. We observed the presence of a band with a 55 kDa apparent molecular weight in the lysates from the VLPs, which was absent in the lysates from the human cell lines (Fig 3B). This finding indicated that mAb 3E4 was binding to a viral component of the VLPs, recognizing a band at an apparent molecular weight of 55 kDa that could be Gag because of the expected migration of p55 Gag. To test our hypothesis, we performed western blot analysis using cell lysates from NL4-3 virus produced in 293T or HeLa cell lines, using mAb 3E4 as a probe. As a positive control, we included capsid-specific mAb CA183 that binds to a region on the top of Gag (Fig 3C, left panel). The results showed that mAb 3E4 binds to p55 Gag and p17 Matrix proteins (Fig 3C, right panel). These findings revealed that the VH4-59 gene-encoded mAbs bind to the p17 Matrix protein.

Bottom Line: We found no evidence that mAb 3E4 exhibited any function in laboratory studies aimed at elucidating the immunologic activity, including assays for neutralization, Ab-dependent cell-mediated virus inhibition, or enhanced T cell reactivity caused by Gag-3E4 complexes.The findings suggest this immunodominant epitope in Gag protein, which is associated with VH4-59 germline gene usage, may induce a high level of B cells that encode binding but non-functional antibodies that occupy significant repertoire space following HIV infection.The studies define an additional specific molecular mechanism in the immune distraction activity of the HIV virion.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Microbiology and Immunology, Vanderbilt University, Nashville, Tennessee, United States of America.

ABSTRACT
The human antibody response against HIV-1 infection recognizes diverse antigenic subunits of the virion, and includes a high level of antibodies to the Gag protein. We report here the isolation and characterization of a subset of Gag-specific human monoclonal antibodies (mAbs) that were prevalent in the antibody repertoire of an HIV-infected individual. Several lineages of Gag-specifc mAbs were encoded by a single antibody heavy chain variable region, VH4-59, and a representative antibody from this group designated mAb 3E4 recognized a linear epitope on the globular head of the p17 subunit of Gag. We found no evidence that mAb 3E4 exhibited any function in laboratory studies aimed at elucidating the immunologic activity, including assays for neutralization, Ab-dependent cell-mediated virus inhibition, or enhanced T cell reactivity caused by Gag-3E4 complexes. The findings suggest this immunodominant epitope in Gag protein, which is associated with VH4-59 germline gene usage, may induce a high level of B cells that encode binding but non-functional antibodies that occupy significant repertoire space following HIV infection. The studies define an additional specific molecular mechanism in the immune distraction activity of the HIV virion.

No MeSH data available.


Related in: MedlinePlus