Mark Melchers - Academia.edu (original) (raw)
Papers by Mark Melchers
Journal of Biological Chemistry, 2015
Many therapeutic proteins and protein subunit vaccines contain heterologous trimerization domains... more Many therapeutic proteins and protein subunit vaccines contain heterologous trimerization domains, such as the widely used GCN4-based isoleucine zipper (IZ) and the T4 bacteriophage fibritin foldon (Fd) trimerization domains. We found that these domains induced potent anti-IZ or anti-Fd antibody responses in animals when fused to an HIV-1 envelope glycoprotein (Env) immunogen. To dampen IZ-induced responses, we constructed an IZ domain containing four N-linked glycans (IZN4) to shield the underlying protein surface. When fused to two different vaccine antigens, HIV-1 Env and influenza hemagglutinin (HA), IZN4 strongly reduced the antibody responses against the IZ, but did not affect the antibody titers against Env or HA. Silencing of immunogenic multimerization domains with glycans might be relevant for therapeutic proteins and protein vaccines.
Trends in Microbiology, 2007
HIV-1 evades antibody-mediated neutralization in many cunning ways. The recent structural charact... more HIV-1 evades antibody-mediated neutralization in many cunning ways. The recent structural characterization of a conserved neutralization epitope on the envelope glycoprotein complex (Env) provides clues to the vulnerabilities of the virus. We discuss these observations and explain their relevance for HIV-1 vaccine design.
Journal of Biological Chemistry, 2010
The envelope glycoproteins (Env) are the focus of HIV-1 vaccine development strategies based on t... more The envelope glycoproteins (Env) are the focus of HIV-1 vaccine development strategies based on the induction of humoral immunity, but the mechanisms the virus has evolved to limit the induction and binding of neutralizing antibodies (NAbs) constitute substantial obstacles. Conserved neutralization epitopes are shielded by variable regions and carbohydrates, so one strategy to increase their exposure and, it is hoped, their immunogenicity is to delete the overlying variable loops. However, deleting the variable regions from Env trimers can be problematic, because hydrophobic patches that are normally solvent-inaccessible now become exposed, causing protein misfolding or aggregation, for example. Here, we describe the construction and characterization of recombinant gp140 trimers lacking variable domains 1 and 2 (⌬V1V2). The design of the trimers was guided by HIV-1 evolution studies that identified compensatory changes in V1V2-deleted but functional Env proteins (We now show that specific compensatory changes improved the function of ⌬V1V2 Env proteins and hence HIV-1 replication. The changes acted by reducing the exposure of a hydrophobic surface either by replacing a hydrophobic residue with a hydrophilic one or by covering the surface with a glycan. The compensatory changes allowed the efficient expression of well folded, soluble gp140 trimers derived from various HIV-1 isolates. The evolved ⌬V1V2 Env viruses were extremely sensitive to NAbs, indicating that neutralization epitopes are well exposed, which was confirmed by studies of NAb binding to the soluble ⌬V1V2 gp140 trimers. These evolved ⌬V1V2 trimers could be useful reagents for immunogenicity and structural studies. . The abbreviations used are: Env, envelope glycoproteins complex; NAb, neutralizing antibody; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; Ni-NTA, nickel-nitrilotriacetic acid; GnTI, N-acetylglucosaminyltransferase I.
Vaccines, 2013
HIV-1 DNA vaccines have many advantageous features. Evaluation of HIV-1 vaccine candidates often ... more HIV-1 DNA vaccines have many advantageous features. Evaluation of HIV-1 vaccine candidates often starts in small animal models before macaque and human trials. Here, we selected and optimized DNA vaccine candidates through systematic testing in rabbits for the induction of broadly neutralizing antibodies (bNAb). We compared three
Interferon (IFN)
CORRECTION < d o c h e a d > C o r r e c t i o n < / d o c h e a d > < d o c t o p i c > C o r r ... more CORRECTION < d o c h e a d > C o r r e c t i o n < / d o c h e a d > < d o c t o p i c > C o r r e c t i o n s < / d o c t o p i c > < d o i > d o i : 1 0 . 1 0 8 4 / j e m . 2 0 0 5 1 4 5 0 0 1 2 5 0 6 c < /
Virology, 2010
The HIV-1 envelope glycoprotein complex (Env) is the focus of vaccine development aimed at elicit... more The HIV-1 envelope glycoprotein complex (Env) is the focus of vaccine development aimed at eliciting humoral immunity. Env's extensive and heterogeneous N-linked glycosylation affects folding, binding to lectin receptors, antigenicity and immunogenicity. We characterized recombinant Env proteins and virus particles produced in mammalian cells that lack N-acetylglucosaminyltransferase I (GnTI), an enzyme necessary for the conversion of oligomannose N-glycans to complex N-glycans. Carbohydrate analyses revealed that trimeric Env produced in GnTI -/cells contained exclusively oligomannose N-glycans, with incompletely trimmed oligomannose glycans predominating. The folding and conformation of Env proteins was little affected by the manipulation of the glycosylation. Viruses produced in GnTI -/cells were infectious, indicating that the conversion to complex glycans is not necessary for Env entry function, although virus binding to the C-type lectin DC-SIGN was enhanced. Manipulating Env's N-glycosylation may be useful for structural and functional studies and for vaccine design.
Virology, 2009
Many viruses transmitted via the genital or oral mucosa have the potential to interact with dendr... more Many viruses transmitted via the genital or oral mucosa have the potential to interact with dendritic cellspecific intercellular adhesion molecule-3 grabbing non integrin (DC-SIGN) expressed on immature dendritic cells (iDCs) that lie below the mucosal surface. These cells have been postulated to capture and disseminate human immunodeficiency virus type-1 (HIV-1) to CD4 + lymphocytes, potentially through breaches in the mucosal lining. We have previously described that BSSL (bile salt-stimulated lipase) in human milk can bind DC-SIGN and block transfer. Here we demonstrate that seminal plasma has similar DC-SIGN blocking properties as BSSL in human milk. Using comparative SDS-PAGE and Western blotting combined with mass spectrometry we identified mucin 6 as the DC-SIGN binding component in seminal plasma. Additionally, we demonstrate that purified mucin 6 binds DC-SIGN and successfully inhibits viral transfer. Mucin 6 in seminal plasma may therefore interfere with the sexual transmission of HIV-1 and other DC-SIGN co-opting viruses.
![Research paper thumbnail of Corrigendum to “Lack of complex N-glycans on HIV-1 envelope glycoproteins preserves protein conformation and entry function” [Virology 401 (2010) 236–247]](https://attachments.academia-assets.com/48884099/thumbnails/1.jpg)
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Retrovirology, 2008
The HIV-1 envelope glycoprotein gp120, which mediates viral attachment to target cells, consists ... more The HIV-1 envelope glycoprotein gp120, which mediates viral attachment to target cells, consists for ~50% of sugar, but the role of the individual sugar chains in various aspects of gp120 folding and function is poorly understood. Here we studied the role of the carbohydrate at position 386. We identified a virus variant that had lost the 386 glycan in an evolution study of a mutant virus lacking the disulfide bond at the base of the V4 domain.
Retrovirology, 2011
The development of an HIV-1 vaccine that elicits strong neutralizing antibody (nAb) and T cell re... more The development of an HIV-1 vaccine that elicits strong neutralizing antibody (nAb) and T cell responses is challenging. Classical vaccine strategies such as live attenuated vaccines are considered unsafe whereas envelope glycoprotein (Env)subunit vaccines induce low nAb titers that do not protect against HIV-1 infection. We showed previously that most HIV-1-antibody immune complexes (HIV-ICs) formed with either broadly nAbs or Abs derived from patient sera dissociate into free HIV-1 virions and Ab when captured by dendritic cells (DCs). Dissociation of HIV-ICs allows for transmission from DCs to CD4 + T target cells. H but more importantly it can hamper the activation of immune cells which is a hallmark of stable ICs. The natural role of ICs is enhancing uptake by DCs, DC activation, induction of antigen presentation and induction of T cell responses. Furthermore, ICs are captured by follicular DCs that activate the B cells for Ab production, Ab affinity maturation and ísotype switching. We explore stable Env-ICs as a vaccine candidate. To form stable Env-ICs we fused the Fc-region of immunoglobulins to trimeric gp140. Env-IC maintained a native Env conformation which was evaluated by ELISA with Env-specific Abs. Native PAGE analyses and size exclusion chromatography showed that Env-ICs formed trimers, but hexamers consisting of 2 Env trimers and 3 dimeric Fc-tails were also observed. The functionality of the Fc-tail was evaluated by immuno-precipitation of the Env-IC with protein-G couple beads. Capture of Env-IC by DCs was enhanced with 50% compared to wild-type Env. Moreover, Env-IC captured by DCs more efficiently activated gp120-specificT helper cells.
PLoS ONE, 2013
Despite almost 30 years of research, no effective vaccine has yet been developed against HIV-1. P... more Despite almost 30 years of research, no effective vaccine has yet been developed against HIV-1. Probably such a vaccine would need to induce both an effective T cell and antibody response. Any vaccine component focused on inducing humoral immunity requires the HIV-1 envelope (Env) glycoprotein complex as it is the only viral protein exposed on the virion surface. HIV-1 has evolved several mechanisms to evade broadly reactive neutralizing antibodies. One such a mechanism involves variable loop domains, which are highly flexible structures that shield the underlying conserved epitopes. We hypothesized that removal of such loops would increase the exposure and immunogenicity of these conserved regions. Env variable loop deletion however often leads to protein misfolding and aggregation because hydrophobic patches becoming solvent accessible. We have therefore previously used virus evolution to acquire functional Env proteins lacking the V1V2 loop. We then expressed them in soluble (uncleaved) gp140 forms. Three mutants were found to perform optimally in terms of protein expression, stability, trimerization and folding. In this study, we characterized the immune responses to these antigens in rabbits. The V1V2 deletion mutant DV1V2.9.VK induced a prominent response directed to epitopes that are not fully available on the other Env proteins tested but that effectively bound and neutralized the DV1V2 Env virus. This Env variant also induced more efficient neutralization of the tier 1 virus SF162. The immune refocusing effect was lost after booster immunization with a full-length gp140 protein with intact V1V2 loops. Collectively, this result suggests that deletion of variable domains could alter the specificity of the humoral immune response, but did not result in broad neutralization of neutralization-resistant virus isolates.
Molecular Immunology, 2007
Herpesviruses employ many mechanisms to evade the immune response, allowing them to persist life-... more Herpesviruses employ many mechanisms to evade the immune response, allowing them to persist life-long in their hosts. The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) and, more recently, the latency-associated nuclear antigen 1 (LANA-1) of the Kaposi Sarcoma Herpesvirus have been shown to function as in cis-acting inhibitors of antigen presentation. In both proteins, long simple repeat elements are responsible for the inhibition, but the sequences of these repeats are strongly dissimilar. Intriguingly, EBNA-1 mRNA contains a large nested open reading frame that codes for a 40.7kDa strongly acidic protein, in addition to the full-length EBNA-1. This protein, here called pGZr, has a 230 amino-acids long glycine, glutamine, and glutamic acid-rich repeat (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;GZ&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; repeat), highly similar (65% amino-acid identity) to the acidic repeat of LANA-1. To evaluate if pGZr, like EBNA-1 and LANA-1, can inhibit antigen presentation in cis, we fused the nested ORF with the E. coli-derived LacZ gene encoding beta-galactosidase. Whereas cells producing the unmodified beta-galactosidase readily present the H-2L(d)-restricted CTL epitope TPHPARIGL, which resides in the C-terminal region of beta-galactosidase, cells producing the pGZr-beta-galactosidase fusion protein do not. Also shorter fragments of the repeat can inhibit peptide presentation. Even though the physiological function of pGZr remains to be elucidated, the GZ-repeat protein may be valuable as inhibitor of presentation of antigenic peptides derived from transgenes in gene therapy.
Journal of Virology, 2012
An HIV-1 vaccine remains elusive, in part because various factors limit the quantity and quality ... more An HIV-1 vaccine remains elusive, in part because various factors limit the quantity and quality of the antibodies raised against the viral envelope glycoprotein complex (Env). We hypothesized that targeting Env vaccines directly to B cells, by fusing them to molecules that bind and activate these cells, would improve Env-specific antibody responses. Therefore, we fused trimeric Env gp140 to A PRoliferation-Inducing Ligand (APRIL), B-cell Activating Factor (BAFF), and CD40 Ligand (CD40L). The Env-APRIL, Env-BAFF, and Env-CD40L gp140 trimers all enhanced the expression of activation-induced cytidine deaminase (AID), the enzyme responsible for inducing somatic hypermutation, antibody affinity maturation, and antibody class switching. They also triggered IgM, IgG, and IgA secretion from human B cells in vitro. The Env-APRIL trimers induced higher anti-Env antibody responses in rabbits, including neutralizing antibodies against tier 1 viruses. The enhanced Env-specific responses were not associated with a general increase in total plasma antibody concentrations, indicating that the effect of APRIL was specific for Env. All the rabbit sera raised against gp140 trimers, irrespective of the presence of CD40L, BAFF, or APRIL, recognized trimeric Env efficiently, whereas sera raised against gp120 monomers did not. The levels of trimer-binding and virus-neutralizing antibodies were strongly correlated, suggesting that gp140 trimers are superior to gp120 monomers as immunogens. Targeting and activating B cells with a trimeric HIV-1 Env-APRIL fusion protein may therefore improve the induction of humoral immunity against HIV-1.
Journal of Experimental Medicine, 2008
Journal of Biological Chemistry, 2015
Many therapeutic proteins and protein subunit vaccines contain heterologous trimerization domains... more Many therapeutic proteins and protein subunit vaccines contain heterologous trimerization domains, such as the widely used GCN4-based isoleucine zipper (IZ) and the T4 bacteriophage fibritin foldon (Fd) trimerization domains. We found that these domains induced potent anti-IZ or anti-Fd antibody responses in animals when fused to an HIV-1 envelope glycoprotein (Env) immunogen. To dampen IZ-induced responses, we constructed an IZ domain containing four N-linked glycans (IZN4) to shield the underlying protein surface. When fused to two different vaccine antigens, HIV-1 Env and influenza hemagglutinin (HA), IZN4 strongly reduced the antibody responses against the IZ, but did not affect the antibody titers against Env or HA. Silencing of immunogenic multimerization domains with glycans might be relevant for therapeutic proteins and protein vaccines.
Trends in Microbiology, 2007
HIV-1 evades antibody-mediated neutralization in many cunning ways. The recent structural charact... more HIV-1 evades antibody-mediated neutralization in many cunning ways. The recent structural characterization of a conserved neutralization epitope on the envelope glycoprotein complex (Env) provides clues to the vulnerabilities of the virus. We discuss these observations and explain their relevance for HIV-1 vaccine design.
Journal of Biological Chemistry, 2010
The envelope glycoproteins (Env) are the focus of HIV-1 vaccine development strategies based on t... more The envelope glycoproteins (Env) are the focus of HIV-1 vaccine development strategies based on the induction of humoral immunity, but the mechanisms the virus has evolved to limit the induction and binding of neutralizing antibodies (NAbs) constitute substantial obstacles. Conserved neutralization epitopes are shielded by variable regions and carbohydrates, so one strategy to increase their exposure and, it is hoped, their immunogenicity is to delete the overlying variable loops. However, deleting the variable regions from Env trimers can be problematic, because hydrophobic patches that are normally solvent-inaccessible now become exposed, causing protein misfolding or aggregation, for example. Here, we describe the construction and characterization of recombinant gp140 trimers lacking variable domains 1 and 2 (⌬V1V2). The design of the trimers was guided by HIV-1 evolution studies that identified compensatory changes in V1V2-deleted but functional Env proteins (We now show that specific compensatory changes improved the function of ⌬V1V2 Env proteins and hence HIV-1 replication. The changes acted by reducing the exposure of a hydrophobic surface either by replacing a hydrophobic residue with a hydrophilic one or by covering the surface with a glycan. The compensatory changes allowed the efficient expression of well folded, soluble gp140 trimers derived from various HIV-1 isolates. The evolved ⌬V1V2 Env viruses were extremely sensitive to NAbs, indicating that neutralization epitopes are well exposed, which was confirmed by studies of NAb binding to the soluble ⌬V1V2 gp140 trimers. These evolved ⌬V1V2 trimers could be useful reagents for immunogenicity and structural studies. . The abbreviations used are: Env, envelope glycoproteins complex; NAb, neutralizing antibody; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; Ni-NTA, nickel-nitrilotriacetic acid; GnTI, N-acetylglucosaminyltransferase I.
Vaccines, 2013
HIV-1 DNA vaccines have many advantageous features. Evaluation of HIV-1 vaccine candidates often ... more HIV-1 DNA vaccines have many advantageous features. Evaluation of HIV-1 vaccine candidates often starts in small animal models before macaque and human trials. Here, we selected and optimized DNA vaccine candidates through systematic testing in rabbits for the induction of broadly neutralizing antibodies (bNAb). We compared three
Interferon (IFN)
CORRECTION < d o c h e a d > C o r r e c t i o n < / d o c h e a d > < d o c t o p i c > C o r r ... more CORRECTION < d o c h e a d > C o r r e c t i o n < / d o c h e a d > < d o c t o p i c > C o r r e c t i o n s < / d o c t o p i c > < d o i > d o i : 1 0 . 1 0 8 4 / j e m . 2 0 0 5 1 4 5 0 0 1 2 5 0 6 c < /
Virology, 2010
The HIV-1 envelope glycoprotein complex (Env) is the focus of vaccine development aimed at elicit... more The HIV-1 envelope glycoprotein complex (Env) is the focus of vaccine development aimed at eliciting humoral immunity. Env's extensive and heterogeneous N-linked glycosylation affects folding, binding to lectin receptors, antigenicity and immunogenicity. We characterized recombinant Env proteins and virus particles produced in mammalian cells that lack N-acetylglucosaminyltransferase I (GnTI), an enzyme necessary for the conversion of oligomannose N-glycans to complex N-glycans. Carbohydrate analyses revealed that trimeric Env produced in GnTI -/cells contained exclusively oligomannose N-glycans, with incompletely trimmed oligomannose glycans predominating. The folding and conformation of Env proteins was little affected by the manipulation of the glycosylation. Viruses produced in GnTI -/cells were infectious, indicating that the conversion to complex glycans is not necessary for Env entry function, although virus binding to the C-type lectin DC-SIGN was enhanced. Manipulating Env's N-glycosylation may be useful for structural and functional studies and for vaccine design.
Virology, 2009
Many viruses transmitted via the genital or oral mucosa have the potential to interact with dendr... more Many viruses transmitted via the genital or oral mucosa have the potential to interact with dendritic cellspecific intercellular adhesion molecule-3 grabbing non integrin (DC-SIGN) expressed on immature dendritic cells (iDCs) that lie below the mucosal surface. These cells have been postulated to capture and disseminate human immunodeficiency virus type-1 (HIV-1) to CD4 + lymphocytes, potentially through breaches in the mucosal lining. We have previously described that BSSL (bile salt-stimulated lipase) in human milk can bind DC-SIGN and block transfer. Here we demonstrate that seminal plasma has similar DC-SIGN blocking properties as BSSL in human milk. Using comparative SDS-PAGE and Western blotting combined with mass spectrometry we identified mucin 6 as the DC-SIGN binding component in seminal plasma. Additionally, we demonstrate that purified mucin 6 binds DC-SIGN and successfully inhibits viral transfer. Mucin 6 in seminal plasma may therefore interfere with the sexual transmission of HIV-1 and other DC-SIGN co-opting viruses.
Retrovirology, 2008
The HIV-1 envelope glycoprotein gp120, which mediates viral attachment to target cells, consists ... more The HIV-1 envelope glycoprotein gp120, which mediates viral attachment to target cells, consists for ~50% of sugar, but the role of the individual sugar chains in various aspects of gp120 folding and function is poorly understood. Here we studied the role of the carbohydrate at position 386. We identified a virus variant that had lost the 386 glycan in an evolution study of a mutant virus lacking the disulfide bond at the base of the V4 domain.
Retrovirology, 2011
The development of an HIV-1 vaccine that elicits strong neutralizing antibody (nAb) and T cell re... more The development of an HIV-1 vaccine that elicits strong neutralizing antibody (nAb) and T cell responses is challenging. Classical vaccine strategies such as live attenuated vaccines are considered unsafe whereas envelope glycoprotein (Env)subunit vaccines induce low nAb titers that do not protect against HIV-1 infection. We showed previously that most HIV-1-antibody immune complexes (HIV-ICs) formed with either broadly nAbs or Abs derived from patient sera dissociate into free HIV-1 virions and Ab when captured by dendritic cells (DCs). Dissociation of HIV-ICs allows for transmission from DCs to CD4 + T target cells. H but more importantly it can hamper the activation of immune cells which is a hallmark of stable ICs. The natural role of ICs is enhancing uptake by DCs, DC activation, induction of antigen presentation and induction of T cell responses. Furthermore, ICs are captured by follicular DCs that activate the B cells for Ab production, Ab affinity maturation and ísotype switching. We explore stable Env-ICs as a vaccine candidate. To form stable Env-ICs we fused the Fc-region of immunoglobulins to trimeric gp140. Env-IC maintained a native Env conformation which was evaluated by ELISA with Env-specific Abs. Native PAGE analyses and size exclusion chromatography showed that Env-ICs formed trimers, but hexamers consisting of 2 Env trimers and 3 dimeric Fc-tails were also observed. The functionality of the Fc-tail was evaluated by immuno-precipitation of the Env-IC with protein-G couple beads. Capture of Env-IC by DCs was enhanced with 50% compared to wild-type Env. Moreover, Env-IC captured by DCs more efficiently activated gp120-specificT helper cells.
PLoS ONE, 2013
Despite almost 30 years of research, no effective vaccine has yet been developed against HIV-1. P... more Despite almost 30 years of research, no effective vaccine has yet been developed against HIV-1. Probably such a vaccine would need to induce both an effective T cell and antibody response. Any vaccine component focused on inducing humoral immunity requires the HIV-1 envelope (Env) glycoprotein complex as it is the only viral protein exposed on the virion surface. HIV-1 has evolved several mechanisms to evade broadly reactive neutralizing antibodies. One such a mechanism involves variable loop domains, which are highly flexible structures that shield the underlying conserved epitopes. We hypothesized that removal of such loops would increase the exposure and immunogenicity of these conserved regions. Env variable loop deletion however often leads to protein misfolding and aggregation because hydrophobic patches becoming solvent accessible. We have therefore previously used virus evolution to acquire functional Env proteins lacking the V1V2 loop. We then expressed them in soluble (uncleaved) gp140 forms. Three mutants were found to perform optimally in terms of protein expression, stability, trimerization and folding. In this study, we characterized the immune responses to these antigens in rabbits. The V1V2 deletion mutant DV1V2.9.VK induced a prominent response directed to epitopes that are not fully available on the other Env proteins tested but that effectively bound and neutralized the DV1V2 Env virus. This Env variant also induced more efficient neutralization of the tier 1 virus SF162. The immune refocusing effect was lost after booster immunization with a full-length gp140 protein with intact V1V2 loops. Collectively, this result suggests that deletion of variable domains could alter the specificity of the humoral immune response, but did not result in broad neutralization of neutralization-resistant virus isolates.
Molecular Immunology, 2007
Herpesviruses employ many mechanisms to evade the immune response, allowing them to persist life-... more Herpesviruses employ many mechanisms to evade the immune response, allowing them to persist life-long in their hosts. The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) and, more recently, the latency-associated nuclear antigen 1 (LANA-1) of the Kaposi Sarcoma Herpesvirus have been shown to function as in cis-acting inhibitors of antigen presentation. In both proteins, long simple repeat elements are responsible for the inhibition, but the sequences of these repeats are strongly dissimilar. Intriguingly, EBNA-1 mRNA contains a large nested open reading frame that codes for a 40.7kDa strongly acidic protein, in addition to the full-length EBNA-1. This protein, here called pGZr, has a 230 amino-acids long glycine, glutamine, and glutamic acid-rich repeat (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;GZ&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; repeat), highly similar (65% amino-acid identity) to the acidic repeat of LANA-1. To evaluate if pGZr, like EBNA-1 and LANA-1, can inhibit antigen presentation in cis, we fused the nested ORF with the E. coli-derived LacZ gene encoding beta-galactosidase. Whereas cells producing the unmodified beta-galactosidase readily present the H-2L(d)-restricted CTL epitope TPHPARIGL, which resides in the C-terminal region of beta-galactosidase, cells producing the pGZr-beta-galactosidase fusion protein do not. Also shorter fragments of the repeat can inhibit peptide presentation. Even though the physiological function of pGZr remains to be elucidated, the GZ-repeat protein may be valuable as inhibitor of presentation of antigenic peptides derived from transgenes in gene therapy.
Journal of Virology, 2012
An HIV-1 vaccine remains elusive, in part because various factors limit the quantity and quality ... more An HIV-1 vaccine remains elusive, in part because various factors limit the quantity and quality of the antibodies raised against the viral envelope glycoprotein complex (Env). We hypothesized that targeting Env vaccines directly to B cells, by fusing them to molecules that bind and activate these cells, would improve Env-specific antibody responses. Therefore, we fused trimeric Env gp140 to A PRoliferation-Inducing Ligand (APRIL), B-cell Activating Factor (BAFF), and CD40 Ligand (CD40L). The Env-APRIL, Env-BAFF, and Env-CD40L gp140 trimers all enhanced the expression of activation-induced cytidine deaminase (AID), the enzyme responsible for inducing somatic hypermutation, antibody affinity maturation, and antibody class switching. They also triggered IgM, IgG, and IgA secretion from human B cells in vitro. The Env-APRIL trimers induced higher anti-Env antibody responses in rabbits, including neutralizing antibodies against tier 1 viruses. The enhanced Env-specific responses were not associated with a general increase in total plasma antibody concentrations, indicating that the effect of APRIL was specific for Env. All the rabbit sera raised against gp140 trimers, irrespective of the presence of CD40L, BAFF, or APRIL, recognized trimeric Env efficiently, whereas sera raised against gp120 monomers did not. The levels of trimer-binding and virus-neutralizing antibodies were strongly correlated, suggesting that gp140 trimers are superior to gp120 monomers as immunogens. Targeting and activating B cells with a trimeric HIV-1 Env-APRIL fusion protein may therefore improve the induction of humoral immunity against HIV-1.
Journal of Experimental Medicine, 2008