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Immunogens Modeling a Fusion-Intermediate Conformation of gp41 Elicit Antibodies to the Membrane Proximal External Region of the HIV Envelope Glycoprotein.

Vassell R, He Y, Vennakalanti P, Dey AK, Zhuang M, Wang W, Sun Y, Biron-Sorek Z, Srivastava IK, LaBranche CC, Montefiori DC, Barnett SW, Weiss CD - PLoS ONE (2015)

Bottom Line: In prime-boost studies, the best MPER responses were seen in the groups that received DNA priming with gp41 vectors followed by gp41 protein boosts.Finally, although titers to the entire protein immunogen were similar in the two immunization protocols, MPER-specific titers differed, suggesting that the immunization route, schedule, dose, or adjuvant may differentially influence MPER immunogenicity.These findings inform the design of future MPER immunogens and immunization protocols.

View Article: PubMed Central - PubMed

Affiliation: Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, United States of America.

ABSTRACT
The membrane proximal external region (MPER) of the gp41 subunit of the HIV-1 envelope glycoprotein (Env) contains determinants for broadly neutralizing antibodies and has remained an important focus of vaccine design. However, creating an immunogen that elicits broadly neutralizing antibodies to this region has proven difficult in part due to the relative inaccessibility of the MPER in the native conformation of Env. Here, we describe the antigenicity and immunogenicity of a panel of oligomeric gp41 immunogens designed to model a fusion-intermediate conformation of Env in order to enhance MPER exposure in a relevant conformation. The immunogens contain segments of the gp41 N- and C-heptad repeats to mimic a trapped intermediate, followed by the MPER, with variations that include different N-heptad lengths, insertion of extra epitopes, and varying C-termini. These well-characterized immunogens were evaluated in two different immunization protocols involving gp41 and gp140 proteins, gp41 and gp160 DNA primes, and different immunization schedules and adjuvants. We found that the immunogens designed to reduce extension of helical structure into the MPER elicited the highest MPER antibody binding titers, but these antibodies lacked neutralizing activity. The gp41 protein immunogens also elicited higher MPER titers than the gp140 protein immunogen. In prime-boost studies, the best MPER responses were seen in the groups that received DNA priming with gp41 vectors followed by gp41 protein boosts. Finally, although titers to the entire protein immunogen were similar in the two immunization protocols, MPER-specific titers differed, suggesting that the immunization route, schedule, dose, or adjuvant may differentially influence MPER immunogenicity. These findings inform the design of future MPER immunogens and immunization protocols.

No MeSH data available.


Related in: MedlinePlus

Six-helix bundle structure in the gp41 immunogens.Indirect ELISA of the indicated gp41 immunogens using a monoclonal antibody specific for the six-helix bundle (NC-1)[76]. Positive control is a recombinant six-helix bundle (N34C28). Negative controls are an N-HR peptide (N36), a C-HR peptide (C34) and an irrelevant coiled-coil trimer (A27) which was expressed and purified in a manner similar to the gp41 immunogens [93].
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pone.0128562.g004: Six-helix bundle structure in the gp41 immunogens.Indirect ELISA of the indicated gp41 immunogens using a monoclonal antibody specific for the six-helix bundle (NC-1)[76]. Positive control is a recombinant six-helix bundle (N34C28). Negative controls are an N-HR peptide (N36), a C-HR peptide (C34) and an irrelevant coiled-coil trimer (A27) which was expressed and purified in a manner similar to the gp41 immunogens [93].

Mentions: To determine whether the immunogens folded into a 6HB-like structure as designed, the immunogens were also assessed by circular dichroism spectroscopy. All immunogens displayed high helical content (Fig 3A), and thermal denaturation studies demonstrated that they all had high temperatures for their midpoints of transition (Tm) (Fig 3B), consistent with formation of 6HB structures. The presence of a 6HB structure in the immunogens was further assessed using a monoclonal antibody (NC-1) that is specific for the 6HB [76]. These studies showed that the NC-1 monoclonal binds to the immunogens containing both N- and C-heptad repeat domains (FDA13, FDA18, FDA20, FDA21, and FDA26, FDAB4), but not to immunogens that lack both HR1 and HR2 sequences (A27L recombinant protein or N36 or C34 peptides) (Fig 4). Unexpectedly, NC-1 also bound to FDA22, which has the SIV N-HR and C-HR. This reactivity may reflect cross-reactivity of the monoclonals for the SIV 6HB, however, we cannot rule out the possibility that a heterologous 6HB was formed between the SIV N-HR and the HIV C-HR.


Immunogens Modeling a Fusion-Intermediate Conformation of gp41 Elicit Antibodies to the Membrane Proximal External Region of the HIV Envelope Glycoprotein.

Vassell R, He Y, Vennakalanti P, Dey AK, Zhuang M, Wang W, Sun Y, Biron-Sorek Z, Srivastava IK, LaBranche CC, Montefiori DC, Barnett SW, Weiss CD - PLoS ONE (2015)

Six-helix bundle structure in the gp41 immunogens.Indirect ELISA of the indicated gp41 immunogens using a monoclonal antibody specific for the six-helix bundle (NC-1)[76]. Positive control is a recombinant six-helix bundle (N34C28). Negative controls are an N-HR peptide (N36), a C-HR peptide (C34) and an irrelevant coiled-coil trimer (A27) which was expressed and purified in a manner similar to the gp41 immunogens [93].
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4472232&req=5

pone.0128562.g004: Six-helix bundle structure in the gp41 immunogens.Indirect ELISA of the indicated gp41 immunogens using a monoclonal antibody specific for the six-helix bundle (NC-1)[76]. Positive control is a recombinant six-helix bundle (N34C28). Negative controls are an N-HR peptide (N36), a C-HR peptide (C34) and an irrelevant coiled-coil trimer (A27) which was expressed and purified in a manner similar to the gp41 immunogens [93].
Mentions: To determine whether the immunogens folded into a 6HB-like structure as designed, the immunogens were also assessed by circular dichroism spectroscopy. All immunogens displayed high helical content (Fig 3A), and thermal denaturation studies demonstrated that they all had high temperatures for their midpoints of transition (Tm) (Fig 3B), consistent with formation of 6HB structures. The presence of a 6HB structure in the immunogens was further assessed using a monoclonal antibody (NC-1) that is specific for the 6HB [76]. These studies showed that the NC-1 monoclonal binds to the immunogens containing both N- and C-heptad repeat domains (FDA13, FDA18, FDA20, FDA21, and FDA26, FDAB4), but not to immunogens that lack both HR1 and HR2 sequences (A27L recombinant protein or N36 or C34 peptides) (Fig 4). Unexpectedly, NC-1 also bound to FDA22, which has the SIV N-HR and C-HR. This reactivity may reflect cross-reactivity of the monoclonals for the SIV 6HB, however, we cannot rule out the possibility that a heterologous 6HB was formed between the SIV N-HR and the HIV C-HR.

Bottom Line: In prime-boost studies, the best MPER responses were seen in the groups that received DNA priming with gp41 vectors followed by gp41 protein boosts.Finally, although titers to the entire protein immunogen were similar in the two immunization protocols, MPER-specific titers differed, suggesting that the immunization route, schedule, dose, or adjuvant may differentially influence MPER immunogenicity.These findings inform the design of future MPER immunogens and immunization protocols.

View Article: PubMed Central - PubMed

Affiliation: Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, United States of America.

ABSTRACT
The membrane proximal external region (MPER) of the gp41 subunit of the HIV-1 envelope glycoprotein (Env) contains determinants for broadly neutralizing antibodies and has remained an important focus of vaccine design. However, creating an immunogen that elicits broadly neutralizing antibodies to this region has proven difficult in part due to the relative inaccessibility of the MPER in the native conformation of Env. Here, we describe the antigenicity and immunogenicity of a panel of oligomeric gp41 immunogens designed to model a fusion-intermediate conformation of Env in order to enhance MPER exposure in a relevant conformation. The immunogens contain segments of the gp41 N- and C-heptad repeats to mimic a trapped intermediate, followed by the MPER, with variations that include different N-heptad lengths, insertion of extra epitopes, and varying C-termini. These well-characterized immunogens were evaluated in two different immunization protocols involving gp41 and gp140 proteins, gp41 and gp160 DNA primes, and different immunization schedules and adjuvants. We found that the immunogens designed to reduce extension of helical structure into the MPER elicited the highest MPER antibody binding titers, but these antibodies lacked neutralizing activity. The gp41 protein immunogens also elicited higher MPER titers than the gp140 protein immunogen. In prime-boost studies, the best MPER responses were seen in the groups that received DNA priming with gp41 vectors followed by gp41 protein boosts. Finally, although titers to the entire protein immunogen were similar in the two immunization protocols, MPER-specific titers differed, suggesting that the immunization route, schedule, dose, or adjuvant may differentially influence MPER immunogenicity. These findings inform the design of future MPER immunogens and immunization protocols.

No MeSH data available.


Related in: MedlinePlus